Previous studies in our laboratory have shown that 6-phenylhexyl isothiocyanate (PHITC), enhances N-nitrosomethylbenzylamine (NMBA)-induced esophageal tumorigenesis in F344 rats while the shorter chain analogs, phenylethyl isothiocyanate (PEITC), and 3-phenylpropyl isothiocyanate (PPITC), inhibit NMBA-induced esophageal tumorigenesis. To test the hypothesis that PHITC influences the promotional stage of esophageal tumorigenesis, groups of 22-27 rats were dosed with vehicle or NMBA three times a week for 5 week, and fed a modified AIN-76A diet containing PHITC at concentrations of 0.0, 1.0, and 2.5 micromol/g. At the 25th week, the rats were killed, esophagi harvested and tumors counted. In the groups that received NMBA+PHITC, apparent but statistically insignificant increases in tumor multiplicity of 32 and 42% were found in comparison to rats treated with NMBA alone. A higher frequency of dysplastic lesions was found in rats treated with NMBA+2.5 micromol/g PHITC (71%) when compared to rats treated with NMBA only (12%). To test whether PHITC increased cellular proliferation, we evaluated proliferating cell nuclear antigen (PCNA) expression by immunohistochemistry. While there were no significant increases in PCNA staining in rats treated with NMBA+PHITC compared to rats treated with NMBA only, rats treated with PHITC only had a significantly higher PCNA index compared to untreated controls. Expression of cyclin D1, another biomarker of proliferation, was analyzed by semi-quantitative reverse transcription-polymerase chain reaction. There were no significant increases in cyclin D1 expression in groups treated with NMBA+PHITC compared to the group treated with NMBA only. Thus, while the data suggest a promotional effect by PHITC as manifested by a significant increase in dysplastic leukoplakia by the high dose of PHITC and an increase in the PCNA index by PHITC alone, PHITC does not appear to have a significant effect on esophageal cell proliferation.