It is known that fatty acids (FAs) regulate transcription through a number of FA responsive transcription factors. In order to investigate the effect of FAs on gene regulation in cultured human hepatocytes we examined the effect of palmitate on hepatic glucokinase (GK) promoter activity and expression of transcription factors that regulate GK expression. GK promoter activity was increased in constructs lacking a cAMP response element (CRE), while palmitate incubation decreased GK promoter activity in CRE-negative constructs. Cells exposed to palmitate showed increased levels of PPAR alpha apolipoprotein-AII and -B100 mRNA and decreased levels of SREBP-1c mRNA but there was no effect on LXR alpha and HNF-4 alpha mRNA. In addition, cDNA microarray analysis of short-term (1.5 h) transcriptional regulation by palmitate, oleate, and eicosapentaenoic acid (EPA) identified that oleate and EPA initiated similar changes in the pattern of hepatic gene regulation, whereas palmitate had little effect. (c) 2007 Elsevier Inc. All rights reserved.