The intermediate filament of mature Sertoli cells is vimentin (Vim). One of the toxicological consequences of phthalate exposure is a selective decrease in Vim, an intermediate-sized (10 nm) cytoplasmic microfilament, in Sertoli cells. Vim in Sertoli cells of rats exposed in utero to 100 mg/kg/day di(n-butyl) phthalate (DBP) on gestation days 12-21 was quantified. Immunohistochemical analysis revealed that Vim aggregated in Sertoli cells, but desmin filaments did not. Vim images were extracted from electron microscopic images using the computer program Imaris (Bitplane Scientific, Zeiss) and analyzed using Image-Pro plus (Media Cybernetics, USA). The amount of perinuclear Vim located within 0.5 μm of the nuclear membrane, where most Vim is aggregated, and the Vim volume ratios of the DBP group were similar to those of the vehicle group at 7 and 9 weeks, but those of the DBP group had decreased 0.63-times at 14 weeks and 0.48-times at 17 weeks compared to those of the vehicle groups. The present study showed that the testicular toxicity of in utero exposure to DBP seemed to be delayed type toxicity, and showed that improved morphometric methods cold be used widely for quantitative analysis of cellular cytoplasmic filaments.