Trimethylpentane (TMP) produces nephrotoxicity in male but not in female rats. The toxicity is characterized by an increase in protein droplets in proximal convoluted tubular cells and an increase in the renal concentration of the male-rat-specific protein alpha 2u-globulin. Subcellular fractionation of the kidneys from male rats 24 hr after [3H]TMP administration showed that about 60% of the radiolabeled material was localized in the 116,000g supernatant. Column chromatography of this supernatant resolved the radioactivity into two components; one, which contained about 26% of the radiolabel, coeluted with alpha 2u-globulin and cross-reacted with an antibody specific for alpha 2u-globulin. The remaining component eluted in the low-molecular-weight range (less than 1000 Da) and was assumed to be TMP metabolites. Radiolabel from [3H]TMP in male rat urine also resolved into two components with about 0.1% of the radiolabel in urine coeluting with the alpha 2u-globulin-containing fraction. Radiolabel from TMP in male rat liver 116,000g supernatant and plasma and in female rat kidney 116,000g supernatant eluted as a single component in the low-molecular-weight range. Dialysis (1000-Da cutoff) of male kidney 116,000g supernatant led to a loss of the low-molecular-weight components, but nondialyzable radiolabel (about 20%) still coeluted with the alpha 2u-globulin after gel chromatography. Dialysis against 0.1% sodium dodecyl sulfate led to a loss of both the low- and high-molecular-weight radioactive material. These results suggested that the high-molecular-weight radioactive material was formed by the reversible binding of a radioactive component of TMP to a male-rat-specific protein. Gas chromatography-mass spectrometry of an ethyl acetate extract of the alpha 2u-globulin-containing fractions of TMP-treated male rat kidney 116,000g supernatant identified 2,4,4-trimethyl-2-pentanol as the only bound metabolite to alpha 2u-globulin. These studies provide the first evidence for a reversible binding between a metabolite of TMP and a male-rat-specific protein in the kidney and thus provide important insight delineating a potential mechanism of hydrocarbon-induced hyaline-droplet nephropathy.