Aflatoxins (AFs) are secondary metabolites produced mainly by Aspergillus parasiticus and A. flavus. To study AF regulation, previously isolated non-toxigenic A. parasiticus sec- (for secondary metabolism minus) variants were genetically analysed. In parasexual crossing, the sec-strains failed to form heterokaryons and diploids with other sec-strains. Heterokaryon test results suggested that involvement of cytoplasmic elements in the formation of sec-phenotype was unlikely. At the molecular level, the coding sequence of the sec-aflR (the only known positive regulator of AF pathway) was identical to that of their toxigenic sec+(for secondary metabolism plus) parents. However, the sec-aflR expression was 5- to 10-fold lower compared to that in the sec+forms. RT-PCR analysis demonstrated that the AF pathway genes were expressed in the sec-forms but in trace amounts and in their unprocessed forms. Combined, these results suggest that aflR is necessary but not sufficient for AF production and that elements involved in fungal development directly or indirectly influence its proper function.