We have previously reported induction of fatty acid synthase (FAS) gene expression by insulin and adipocyte differentiation in 3T3-L1 cells. In order to identify sequences responsible for insulin regulation of the FAS gene, chimaeric constructs containing serial deletions of the 5'-flanking region of the rat FAS gene ligated to the chloramphenicol acetyltransferase (CAT) reporter gene were prepared and transfected into 3T3-L1 cells. Plasmids containing 2100 (-2100CAT), 1400 (-1400CAT), 1009 (-1009CAT) and 332 (-332CAT) bp of FAS 5' flanking sequences exhibited comparable basal CAT activities in 3T3-L1 preadipocytes. This activity was 3-fold higher when these constructs were transiently transfected into 3T3-L1 adipocytes. Stably transfected 3T3-L1 cells also exhibited a 3-fold increase in CAT activity upon adipocyte differentiation, indicating that sequences required for the differentiation-dependent increase in FAS expression are located within the 332 bp promoter. Treatment with 10 nM insulin increased CAT activity by 2.1 ± 0.2-, 2.6 ± 0.1-, 2.0 ± 0.2- and 1.7 ± 0.2-fold respectively in 3T3-L1 adipocytes transiently transfected with -2100CAT, -1400CAT, -1009CAT and -332CAT plasmids. CAT activity was increased by 3.0 ± 0.3- and 3.5 ± 0.6-fold respectively by insulin treatment in adipocytes stably transfected with -2100CAT and -1009CAT plasmids. When insulin-responsive H4IIE hepatoma cells were transiently transfected with -2100CAT,-1400CAT, -1009CAT and -332CAT plasmids and then treated with 10 nM insulin, CAT activity increased by 3.1-, 3.1 ± 0.8-, 3.0 ± 0.7- and 2.3 ± 0.5-fold respectively in serum-free media, and by 2.6 ± 0.4-, 3.3 ± 0.9-, 3.1 ± 0.4- and 2.9 ± 0.6-fold respectively in the presence of 0.5 % serum. These results indicate that sequences responsible for insulin regulation of FAS gene are also located within 332 bp of the transcription start site.