Isophorone (CAS No. 78-59-1) was evaluated for the significance of protein binding and associated cellular proliferation in exposed rat preputial glands, the sites of previously documented increased incidence of carcinomas in exposed male rats. Preputial gland and kidney cytosol isolated from Fischer-344 rats (10/sex/dose group) previously administered single 0, 250 and 500 mg/kg doses of 14C-labeled isophorone by oral gavage 24 hours prior to sacrifice revealed isophorone binding primarily to alpha(2u)globulin (alpha(2u)G) of the preputial gland in both males and females and in the kidney in males, but not females. Upon challenge with SDS elution in labeled isophorone-derived cytosol samples, these bonds were readily dissociated. Conversely, extraction of preputial gland and kidney DNA from rats treated with single 500 mg/kg labeled doses yielded no evidence of isophorone binding to DNA, these negative results confirmed in 32P-postlabeling assays with samples from male and female rats treated for 1 week with 500 mg/kg/day isophorone by oral gavage. Preputial gland acinar cells were heavily and comparably (with no dose-related differences) stained immunohistochemically with anti-alpha(2u)G antibody in both male and female groups (8/sex) treated with 0, 250, and 500 mg/kg by oral gavage on 5 days/week for 1 or 4 weeks. Conversely, in the kidneys of males, treatment intensified and extended the duration of alpha(2u)G activity relative to that indicated by limited immunohistochemical staining of proximal tubule cells in the kidneys of controls. Neither control or treated females exhibited any such evidence of alpha(2u)G in the kidney. Using an identical protocol preceded by the administration of BrdU 2 days earlier, rats (8/sex/dose group) were sacrificed for histologic examination of preputial gland and kidney tissues. Immunohistochemical staining with anti-BrdU antibody revealed a significant (Dunnett's test, p < 0.05) increase in cell proliferation in male preputial glands after 1 week's dosing, that was subsequently reduced to a marginal elevation above controls at 4 weeks. A more dramatic (p < 0.01) increase in cell proliferation was noted in male kidneys in association with both 1 and 4 week's dosing. Female preputial glands, but not kidneys, showed non- significant increased cell proliferation. The authors concluded that, isophorone appears not to be genotoxic and the mechanism of documented preputial gland tumorogenicity in association with chronic oral isophorone may be coincidental (based on historic and control variability) rather than a alpha(2u)G-induced pathology and associated hyperplasia, as this association was transient and inconsistent with that in male rat kidneys. In the male rat kidney, isophorone-related increase in alpha(2u)G activity with increased and sustained cell proliferation suggests a mechanism that is not relevent in humans.