Carnitine palmitoyltransferase (CPT)-I catalyses the transfer of long-chain fatty acids from CoA to carnitine for translocation across the mitochondrial inner membrane. Expression of the ‘liver’ isoform of the CPT-I gene ( CPT-I α) is subject to developmental, hormonal and tissue-specific regulation. To understand the basis for control of CPT-I α gene expression, we have characterized the proximal promoter of the CPT-I α gene. Here, we report the sequence of 6839 base pairs of the promoter and the localization of the rat CPT-I α gene to region q43 on chromosome 1. Our studies show that the first 200 base pairs of the promoter are sufficient to drive transcription of the CPT-I α gene. Within this region are two sites that bind both Sp1 and Sp3 transcription factors. In addition, nuclear factor Y (NF-Y) binds the proximal promoter. Mutation at the Sp1 or NF-Y sites severely decreases transcription from the CPT-Iα promoter. Other protein binding sites were identified within the first 200 base pairs of the promoter by DNase I footprinting, and these elements contribute to CPT-I α gene expression. Our studies demonstrate that CPT-I α is a TATA-less gene which utilizes NF-Y and Sp proteins to drive basal expression.