Genes involved in fatty acid degradation (fad) were isolated from Pseudonocardia autotrophica BBRC12444. Six open reading frames and a bi-directional promoter region were identified by DNA sequence analyses and primer extension. The fad gene cluster included five ORFs, designated fadA, fadB, fadR, fadC, and fadD. Base on their amino acid sequence identity, the gene products were identified as acyl-CoA ligase (FadA), enoyl-CoA hydratase (FadB), transcriptional regulator (FadR), cytochrome P450 monooxygenase (FadC), and ferredoxin (FadD). Regulatory protein, FadR, could bind to an operator sequence located in the divergent promoter region between fadR and fadC genes, implicating the control of fatty acid degradation. The real-time quantitative PCR assays revealed that the expression of the fadA, fadB, fadR, and fadC genes was induced by long chain fatty acids and repressed by glucose. All results demonstrated that the fad gene cluster participated in the pathway of the fatty acid catabolism. This is the first bacterial fad gene cluster to be reported.