In biotin-responsive multiple carboxylase deficiency, a characteristic organic aciduria reflects in vivo deficiency of mitochondrial propionyl CoA carboxylase, 3-methylcrotonyl CoA carboxylase, and pyruvate carboxylase. A possible primary or secondary defect in biotin absorption leads to an infantile-onset syndrome, while abnormal holocarboxylase synthetase activity has been identified in the neonatal-onset form. While distinct mitochondrial and cytosolic holocarboxylase synthetase biotinylation systems may exist in avian tissues, the system has not been characterized in humans. Toward this objective, we studied the biotin dependence of a cytosolic carboxylase, acetyl CoA carboxylase (ACC), in cultured skin fibroblasts of both types of multiple carboxylase deficiency. ACC specific activities in control and infantile-onset cells were not distinguishable at all biotin concentrations: with decreasing biotin availability (+ avidin), there were only modest decrements in ACC activity in both these cell types. In contrast, there were pronounced declines of ACC activity in neonatal-onset (holocarboxylase synthetase-deficient) cells after growth in low biotin concentrations, and activity was undetectable in + avidin. ACC activity was rapidly restored with biotin repletion to biotin-starved holocarboxylase synthetase-deficient cells, and this restoration was largely independent of protein synthesis. The behavior of the cytosolic carboxylase, ACC, is in all these respects identical to that of the mitochondrial carboxylases, an observation consistent with the existence of similar biotinylation mechanisms in the two cell compartments. Further, the data support the notion that at least some components of the holocarboxylase synthetase system are shared by mitochondria and cytosol in humans, and are consistent with the suggestion that restoration of activity in biotin-depleted cells represents biotinylation of preexisting enzyme protein. The modest decrements in ACC activity in normal and infantile-onset cells may be related to the compromised epidermal integrity observed in that form of multiple carboxylase deficiency. Finally, ACC and mitochondrial carboxylase activities were compared in cells from mutants representing a spectrum of clinical severity. Cells from later-onset patients of intermediate clinical severity were ultimately classifiable as putative holocarboxylase synthetase-deficient cells on chemical criteria. Accurate etiologic classification cannot be based on clinical presentation alone, and biochemical studies should be performed on all patients. Accordingly, we propose a classification of multiple carboxylase deficiency based on biochemical criteria.