To investigate for the presence of regulator(s) repressing the expression of insulin gene in cells other than pancreatic beta cells, rat insulinoma (RIN) cells secreting insulin were hybridized with fibroblasts from various species. In RIN x L mouse fibroblast hybrids, which maintained most of the parental chromosomes, no insulin transcripts were detected. In three RIN x Indian muntjac fibroblast hybrids and one RIN x human fibroblast hybrid which had lost DNA from the fibroblast parent through subculture, expression of the insulin gene was first extinguished and then reexpressed. This suggests that negative regulator(s), present in fibroblasts, can turn off insulin gene expression in RIN x fibroblast hybrids as long as the gene(s) contributed by the fibroblast are maintained.