Functional and DNA binding analyses were used to investigate transcriptional regulation of liver arginase, a mammalian urea cycle enzyme with marked tissue specificity. Reporter constructs containing the proximal 111 bp of the gene from man and Macaca fascicularis showed over sixfold background activity in HepG2 hepatoma cells, which express significant levels of liver arginase, and 12-fold background activity in minimally expressing HEK cells. Longer constructs, active in both cell lines, showed greater activity in the liver cell line. The constructs showed no activity in arginase-negative NIH 3T3 fibroblasts. A 54-bp dyad insert present in the human sequence and absent in M. fascicularis did not affect function. DNA binding analyses localized multiple liver-specific complexes as well as complexes shared among cell types. Little binding was evident in fibroblast extracts. Despite liver-specific binding, there was no evidence of a strong liver-specific enhancer. HEK and NIH 3T3 nuclear extracts showed strikingly different patterns of DNA binding. These studies demonstrate that molecular regulation of liver arginase transcription is complex and that control mechanisms differ among tissue types.