Objective To study which variables are able to influence women's experience of pain during diagnostic hysteroscopy. Design Multivariate analysis (phase II) after a randomized, controlled trial (phase I). Setting Endoscopic gynecologic center. Patient(s) In phase I, 392 patients were analyzed. Group A: 197 women with carbon dioxide (CO2 ); group B: 195 women with normal saline. In phase II, 392 patients were assigned to two different groups according to their pain experience as measured by a visual analogue scale (VAS): group VAS >3 (170 patients); group VAS ≤3 (222 patients). Intervention(s) Free-anesthesia diagnostic hysteroscopy performed using CO2 or normal saline as distension media. Main Outcome Measure(s) Procedure time, VAS score, image quality, and side effects during and after diagnostic hysteroscopy. Result(s) In phase I the median pain score in group A was 2, whereas in group B it was 3. In phase II the duration of the procedure, nulliparity, and the use of normal saline were significantly correlated with VAS >3. A higher presence of cervical synechiae was observed in the group VAS >3. The multivariate analysis revealed an inverse correlation between parity and a VAS >3, whereas the use of normal saline, the presence of synechiae in the cervical canal, and the duration of the hysteroscopy were all directly correlated to a VAS score >3. Conclusion(s) Pain in hysteroscopy is significantly related to the presence of cervical synechiae, to the duration of the procedure, and to the use of normal saline; conversely, parity seems to have a protective role. Clinical Trial Registration Number NCT01873391.