The use-1 mutation in Salmonella typhimurium confers a complex and pleiotrophic phenotype which is primarily characterized as a temperature-dependent sensitivity to uracil. This sensitivity can be reversed by arginine or citrulline, but not by ornithine, suggesting that the use-1 mutation affects the synthesis or the activity (or both) of carbamoylphosphate synthetase or ornithine carbamoyltransferase (or both). Activity measurements showed that use-1 caused superrepression of both of these enzymes, especially when uracil was present in the medium. Dihydro-orotase and dihydro-orotate oxidase were also superrepressed, but aspartate carbamoyltransferase and orotate phosphoribosyltransferase were not. Lowered nucleotide triphosphate and guanosine tetra- and pentaphosphate pools in use-1 strains indicated that the mutation affected synthesis or breakdown of all of these phosphorylated compounds, but the UTP pool increased by a larger relative factor in use-1 strains in the presence of uracil. The uracil-sensitive phenotype of the use-1 mutation is a complex response to several environmental factors: temperature, aerobiosis, carbon sources, and uracil concentration. Uracil sensitivity was eliminated by alteration of one or more of these factors. Uracil sensitivity was suppressed by several genetic alterations. These include introduction into use-1 strains of a multi-copy ColE1 derivative which carries the structural gene(s) for carbamoylphosphate synthetase, episomes that carry use, mutations including argR and pyrH, and various unclassified intergenic suppressor mutations. These genetic changes increased significantly the expression of carbamoylphosphate synthetase or ornithine carbamoyltransferase (or both). The activity of use-1 is not known, but the facts that it altered expression of at least four unlinked genes (pyrA, pyrC, pyrD, and argI) and that the Escherichia coli F'133 complemented it establish it as a trans-acting regulatory factor.