Biotin deficiency was induced in germ-free rats using three experimental protocols. The results showed the important role of biotin during gestation and suckling. The earlier the deprivation, the earlier the deficiency and the severer the symptoms. In this vitamin deficiency, symptoms occurred that were not observed in the control rats, such as the formation of an intestinal volvulus in rats ingesting an L-isoleucine-supplemented diet. The main biochemical anomalies characteristic of propionic acidemia (ketoacidosis and increased urinary elimination of propionic acid) due to propionyl CoA carboxylase deficiency in man were not observed in the rats that were deprived of biotin for 200 days even after a dietary load of L-isoleucine. Only a small urinary excretion of propionyl-glycine and tiglylglycine was observed. We observed a drop in enzymatic propionyl CoA carboxylase activity in the liver that was proportional to the severity of the vitamin deficiency.