The LIM domain homeobox gene isl-1 was originally isolated by virtue of its ability to bind DNA sequences from the 5'-flanking region of the rat insulin gene. Initial experiments localized isl-1 to the islet cells of the pancreas, suggesting a possible role for this protein in the regulation of insulin gene expression and/or islet cell development. More recent studies have determined that isl-1 is also expressed in the central and peripheral nervous system. We now report that isl-1 gene expression is not restricted to cells of neuroendocrine lineage. Northern blot analysis of rat and hamster fibroblasts, rat keratinocytes, and human epidermoid carcinoma cells detected messenger RNA transcripts that hybridized, at high stringency, to different isl-1 complementary DNA probes. DNA sequence analysis of human and hamster isl-1 complementary DNAs generated by reverse transcription-polymerase chain reaction from nonneuroendocrine cell lines demonstrated 100% conservation of isl-1 amino acid sequences from rat hamster and human species. Western blot analysis with isl-1 antiserum demonstrated that immunoreactive isl-1 was detectable in nuclear extracts from islet cell lines. In contrast, immunoreactive isl-1 was only detected in cytoplasmic extracts from nonneuroendocrine cell lines. The results of these studies demonstrate striking conservation of mammalian isl-1 coding sequences and widespread expression of the isl-1 gene in heterologous cells. These experiments suggest that the biological importance of isl-1 may not be restricted to neuroendocrine cell lineages and raise the possibility that isl-1 function may be regulated by sequestration in distinct cellular compartments.