We describe two children with deficiency of short-chain L-3-hydroxyacyl-CoA dehydrogenase, a new disorder of the mitochondrial beta-oxidation of straight-chain fatty acids. The patients presented with fasting-induced vomiting, and ketosis and low blood glucose, features typical of ketotic hypoglycemia were documented in one. Enzyme assays were performed in cultured skin fibroblasts. In whole fibroblast preparations there was reduced enzyme activity but high residual activity due to the presence of a nonmitochondrial enzyme. In isolated fibroblast mitochondria the residual enzyme activities were 5 and 6% of the normal controls. Activity in an obligate heterozygote was intermediate, suggesting that this is an autosomal recessive disorder.