Objectives: There is considerable interest in the development of an 18F-labeled myocardial perfusion agent, and we are developing 18F-labeled rhodamine dyes to address this significant clinical need. After the preclinical evaluation of several rhodamine derivatives, we found that the 18F-labeled diethylene glycol ester of rhodamine 6G (Rho6G, Fig. 1) shows high uptake in the myocardium and low uptake/rapid clearance from non-target tissues. We previously reported the automated synthesis of Rho6G (J Nucl Med 2014; 55:1252); and now, in preparation for first-in-human studies, we report the results of the microdose toxicology study. Methods: Male and female rats (Sprague Dawley SD) were assigned to two groups, and were administered (non-radioactive) 19F-rhodamine 6G in vehicle (10% v/v ethanol in 0.9% sterile saline) or vehicle alone via intravenous injection in a tail vein at a volume of 0.8 mg/kg, which corresponds to approximately 650 times the expected maximum human dose. The animals were observed for 2 days (Day 3 interim sacrifice) or 14 days (Day 15 terminal sacrifice) to assess possible adverse effects (i.e., mortality, changes in body weight and/or food consumption, injection site reactions, ophthalmic examinations, and clinical and anatomic pathology). Results: On Day 3 of the dosing phase, one female rat died of accidental causes following blood sample collection. All other animals survived to their scheduled sacrifice. No 19F-rhodamine 6G-related clinical observations or ophthalmic findings were noted. No definitive 19F-rhodamine 6G-related alterations in body weight, body weight change, or food consumption were noted, and any changes were inconclusive, sporadic, and/or incidental and nonadverse. Transient erythema was noted at the injection site but this was most likely due to the deep red color of 19F-rhodamine 6G solution and was nonadverse. 19F-rhodamine 6G administration had no effects on hematology, coagulation, or clinical chemistry results. At the interim sacrifice, 19F-rhodamine 6G-related increased heart and pituitary weight occurred in females given 0.8 mg/kg; however, no microscopic correlates were observed for these changes. 19F-rhodamine 6G-related microscopic findings at the intravenous injection site in interim sacrifice animals given 0.8 mg/kg included increased incidences of mixed cell infiltrates and edema. No test article-related organ weight parameters or microscopic findings were noted at the terminal sacrifice. No macroscopic findings were observed at the interim or terminal sacrifice. Conclusion: Intravenous injection of 19F-rhodamine 6G as a single dose at ~650 times the expected human dose was well tolerated in rats: No clinical observations or ophthalmic findings, and no definitive alterations in body weight, body weight change, or food consumption were noted, and no effects on clinical pathology were noted. Nonadverse, transient erythema was noted at the injection site. At the interim sacrifice, 19F-rhodamine 6G-related increased heart and pituitary weight was noted, and mixed cell infiltrates and edema were present at the injection site, none of which were adverse. At the terminal sacrifice, no organ weight changes or microscopic findings were present. No macroscopic findings were noted. Based on these findings, a single intravenous dose of 19F-rhodamine 6G at 0.8 mg/kg was not deemed adverse.