Evaluating immunomodulating effects is currently a fundamental parameter when designing pharmaceutical products. Rather than wait for the appearance of immunotoxic effects in patients, pre-clinical assays that characterize these potential events in experimental models not only can facilitate the understanding of how these phenomena arise, but sometimes reveal findings that can lead to modifications in currently-accepted methodologies used for estimating the pre-clinical assay predictive risk values. Pentamidine isethionate, a drug used as a second option in treating leishmaniasis, has been repeatedly shown in animals and humans to exert a toxicity at the renal, cardiac and hepatic level; however, its immunotoxic effect have not been as fully evaluated. The potential immunomodulatory effects of pentamidine on splenocytes from two species of mice (BALB/c and ICR) were evaluated ex vivo in this study. The results here indicated that there were significant differences in subpopulation profiles between the strains even before treatment with the drug, with the variances most apparent regarding the relative percentages of CD8(+) and CD19(+) cells in splenocyte preparations. The data also showed that the drug preferentially induced toxicity in BALB/c mice CD8(+) and CD19(+) cells more so than in their ICR counterparts. Conversely, the ICR cells seemed to be more susceptible to drug-induced spontaneous blastogenesis than the cells from BALB/c hosts. The differential results seen here confirm that: any potential immunomodulatory effect of a drug should be studied in at least two different mouse strains during an evaluation of overall toxicologic potential; and, there should be attempts to correlate phenotypical findings with those of functionality before a drug can truly be deemed a safe compound.