It has previously been shown that the monoclonal antibody SPM8‐2 recognizes free spermine and spermidine as well as polyamines bound by an amide bond. In the present work it is demonstrated that this antibody also interacts with spermidine, spermine, and to a lesser extent N1‐ and N8‐acetyl spermidine in an ELISA test where the polyamines are bound by reaction with formaldehyde. 3LL Lewis lung carcinoma cells from tumor‐grafted mice were labeled with fluorescein‐conjugated monoclonal antibody SPM8‐2 and analyzed by flow cytometry. Both viable cells and formaldehyde‐fixed and subsequently permeabilized cells showed fluorescent staining. However, most polyamines present in the cells are not directly available for antibody binding. Treatment of fixed cells with DNase or RNase greatly increased fluorescent staining, suggesting that some polyamines are co‐localized with DNA and RNA. Antibody labeling of the cells was prevented by addition of free spermine. 3LL cells from tumors of mice treated by a polyamine depleting regimen had decreased intracellular spermidine levels and bound less antibody when compared to untreated controls. After digestion with RNase, the cells from treated mice bound considerably less fluorescent antibody than tumor cells from untreated mice, while their RNA content was similar. In contrast, fluorescent staining after DNase digestion was only slightly affected by the treatment with a polyamine depleting regimen. This suggests that the pools of polyamines which are co‐localized with RNA are depleted more readily than those associated with DNA. Since only a small proportion of the intracellular polyamines is accessible to the bulky antibodies, treatment with hydrolytic enzymes (DNase, RNase) is necessary to reveal specific compartments of the polyamines and to demonstrate qualitative and semiquantitative differences of their distribution within cells. Cytometry 27:255–261, 1997. © 1997 Wiley‐Liss, Inc.