BALB/cByJ mice have a deficiency of short-chain acyl-CoA dehydrogenase (SCAD), an enzyme of fatty acid beta-oxidation. This mutant mouse strain represents the only animal model for any human inborn error of fatty acid metabolism. We have investigated the molecular basis of this defect by DNA and RNA analyses, comparing these mice with the wild-type predecessor strain BALB/cBy. We found that the mutant strain has a 278-bp deletion in the 3' end of the structural gene for SCAD and reduced steady-state levels of SCAD mRNA. Two major transcripts are produced in the mutant. One contains intronic sequence due to the absence of splicing, and the second transcript results from missplicing of a normal splice donor site to a cryptic splice acceptor site in the 3' terminal exon. Both abnormal transcripts have aberrant stop codons. These results demonstrate the molecular basis of SCAD deficiency in this unique mouse model.