beta-Ketothiolase deficiency is a deficiency in mitochondrial acetoacetyl-CoA thiolase (T2). We present here an update on mutations and polymorphisms in the human T2 gene. No large deletion or insertion has been observed in Southern blot analysis. Seventeen mutations were identified in 13 T2-deficient patients: nine missense, one nonsense, and five splice-site mutations, and two small deletions. Two polymorphic base substitutions were also detected. A common mutation in T2 deficiency has not been detected but 4 mutations (N158D, Q272X, 828 + 1, 1163 + 2) were identified in two independent families. Eleven of 25 mutant alleles identified caused aberrant splicing. In vivo expression analysis of 13 mutant cDNAs using a Lipofectin reagent suggested that T297M, A301P, A380T mutant alleles retain 5-10% normal T2 activity. A correlation between clinical phenotype and genotype in T2 deficiency seems unlikely.