The phyllosilicate clay, hydrated sodium calcium aluminosilicate (HSCAS), has been shown to prevent aflatoxicosis in farm animals by reducing the bioavailability of aflatoxin. The present study was designed to determine the effects of HSCAS on the metabolism of aflatoxin B1 (AFB1) in an aflatoxin-sensitive species. Male Fischer-344 rats were orally dosed with 1.0, 0.5, 0.25 and 0.125 mg AFB1/kg body weight alone and in combination with 0.5% HSCAS. Urine samples were collected after 6, 24, 36, and 48 h. Aflatoxin M1 (AFM1) and aflatoxin P1 (AFP1) were detected in most urine samples, with or without HSCAS. AFM1 was found to be the major metabolite. Metabolite concentrations were significantly decreased in the presence of HSCAS, and more importantly, no additional metabolites were detected. Our results suggest that the AFB1-HSCAS complex was not significantly dissociated in vivo, and support earlier findings that HSCAS tightly binds aflatoxin.