Male B6C3F1 mice were exposed to 2200 of 7000 ppm isoprene by inhalation for 6 months, followed by a 6-month holding period. An increased incidence of harderian gland (HG) neoplasms was observed at the end of the study. The present study was designed to characterize the genetic alterations in the K- and H-ras protooncogenes in paraffin-embedded sections of HG neoplasms. A high frequency of K- and H-ras mutations was detected in isoprene-induced neoplasms (100%), as compared to spontaneous HG neoplasms from control B6C3F1 mice (56%). The predominant mutations consisted of A to T transversions (CAA to CTA) at K-ras codon 61 (15/30) and C to A transverions (CAA to AAA) at H-ras codon 61 (8/30). Enhanced cell proliferation was observed in HG neoplasms with K-and H-ras CTA mutations. These data suggest that activated ras protooncogenes and enhanced cell proliferation contribute to HG tumorigenesis in isoprene-exposed B6C3F1 mice.