CAR1 (arginase) gene expression responds to multiple environmental signals; expression is induced in response to the intracellular accumulation of arginine and repressed when readily transported and catabolized nitrogen sources are available in the environment. Up to 14 cis-acting sites and 9 trans-acting factors have been implicated in regulated CAR1 transcription. In all but one case, the sites are redundant. To test whether these sites actually participate in CAR1 expression, each class of sites was inactivated by substitution mutations that retained the native spacing of the CAR1 cis-acting elements. Three types of sites function independently of the nitrogen source: two clusters of Abflp- and Rap1p-binding sites, and a GC-rich sequence. Two different sets of nitrogen source-dependent sites are also required: the first consists of two GATAA-containing UASNTR sites that mediate nitrogen catabolite repression-sensitive transcription, and the second is arginine dependent and consists of three UAS1 elements that activate transcription only when arginine is present. A single URS1 site mediates repression of CAR1 arginine-independent upstream activator site (UAS) activity in the absence of arginine and the presence of a poor nitrogen source (a condition under which the inducer-independent Gln3p can function in association with the UASNTR sites). When arginine is present, the combined activity of the UAS elements overcomes the negative effects mediated by URS1. Mutation of the classes of sites either singly or in combination markedly alters CAR1 promoter operation and control, supporting the idea that they function synergistically to regulate expression of the gene.