Orotic aciduria (type 1) results from a mutation in the gene for UMP synthase, a bifunctional protein containing the two enzyme activities which convert orotic acid and 5-phosphoribosyl-1-pyrophosphate to UMP and CO2. In fibroblasts from individuals with orotic aciduria, these two enzymatic activities are about 1% of normal but increase dramatically when deficient cells are grown in the presence of 6-azauridine. Using a polyclonal antiserum to sodium dodecyl sulfate-denatured, pure human UMP synthase, we show that fibroblasts from a patient with orotic aciduria have a low level of immunoreactive UMP synthase protein. Pulse-chase analysis reveals that the UMP synthase is degraded rapidly in the deficient cells. Growth of deficient cells in 6-azauridine leads to an increase in UMP synthase protein and its two enzymatic activities via a decreased rate of proteolytic degradation of UMP synthase. UMP synthase in extracts from deficient cells is more readily denatured by heat and is stabilized after growth of cells in 6-azauridine. These data suggest that the detrimental deficiency of this one patient results from a structurally altered UMP synthase that is probably present in low steady-state amounts due to proteolysis and that this labile protein can be stabilized against heat denaturation and proteolytic degradation by 6-aza-UMP.