A 3.7 kilobase fragment of Dictyostelium discoideum genomic DNA has been cloned by its ability to complement a yeast ura3 mutation affecting the activity of orotidine-5'-phosphate carboxy-lyase (EC 18.104.22.168). This fragment also complements a yeast ura5 mutation that leads to a defect in orotate phosphoribosyl transferase (EC 22.214.171.124). The orotidine-5'-phosphate carboxy-lyase and the orotate phosphoribosyl transferase activities that result from Dictyostelium gene expression in yeast have been detected. The size of the DNA required for both complementations has been localised to a segment of less than 2 kb. A unique Dictyostelium RNA species of 1,600 base pairs hybridizes to this fragment. In vitro deletions in this fragment lead to the simultaneous loss of the two activities. The two enzymatic activities coelute as a protein of 120,000 daltons during gel filtration of a Dictyostelium extract. These results favour the existence, on the cloned Dictyostelium DNA fragment, of a unique structural gene which codes for a bifunctional enzyme carrying the two activities, orotidine-5'-phosphate carboxy-lyase and orotate phosphoribosyl transferase.