Liver fatty acid binding protein (L-FABP) is a small protein that is thought to play an important role in the intracellular binding and trafficking of long chain fatty acids in the liver. Expression of the gene encoding the zebrafish liver fatty acid binding protein is regulated by a 435-bp distal region (-1944 to -1510) of the L-FABP promoter. The 435-bp sequence is sufficient for gene activation in the liver primordia (or bud) and continues to be active in the adult liver when positioned adjacent to the SV40 basal promoter and linked directly to green fluorescent protein. The 435-bp sequence region has two distinct liver regulatory elements, A (-1944 to -1623) and B (-1622 to -1510), and contains multiple putative consensus binding sites. The element A sequence includes two consensus HFH and one HNF-1α site and the element B sequence includes one consensus HNF-3β site. Deletion of an internal 435-bp fragment (-1944 to -1510) including the A and B elements totally ablated the liver-specific activity of the zebrafish L-FABP gene promoter. Deletion of either of the two elements reduces the liver activity. Mutation of the HNF-1α site or either of the two HFH sites in the A element or the HNF-3β site in the B element significantly altered specificity in the liver primordia of transient expression embryos. The importance of the HNF-1α consensus binding site in the A element and the HNF-3β consensus binding site in the B element within the 435-bp distal region of the L-FABP promoter region suggests that combinatorial interactions between multiple regulatory factors are responsible for the gene expression of L-FABP in the liver.