Chronic exposure to MIBK caused an increased incidence of renal tubule adenomas and occurrence of renal tubule carcinomas in male, but not female rats. A number of chemicals have been shown to cause male rat renal tumors through the α2u nephropathy-mediated mode of action (MOA). The objective of this study was to evaluate the ability of MIBK to induce measures of α2u nephropathy in male and female F344 rats following exposure to the same inhalation concentrations used in the cancer bioassay (0, 450, 900, or 1800 ppm). Rats were exposed 6 hours/day for 1 or 4 weeks and kidneys excised approximately 18 hours post exposure to evaluate hyaline droplet accumulation (HDA), α2u staining of hyaline droplets, renal cell proliferation, and to quantitate renal α2u concentration at both 1 and 4 weeks. There was an exposure-related increase in all measures of α2u nephropathy in the male, but not female rat kidneys. The hyaline droplets present in the male rat kidney stained positively for α2u. The changes in HDA and α2u concentration were comparable to d-limonene, an acknowledged inducer of α2u nephropathy. In a separate in vitro investigation using a two-compartment vial equilibration model to assess the interaction between MIBK and α2u, the dissociation constant (Kd) was estimated to be 1.27×10-5M. This Kd is within the range similar to other chemicals known to bind to α2u and cause nephropathy. Together, the exposure-related increase in measures of α2u nephropathy, sustained increase in renal cell proliferation along with an indication of reversible binding of MIBK to α2u, support the inclusion of MIBK in the category of chemicals exerting renal effects through a protein droplet α2u nephropathy-mediated MOA.