L-CAM is a calcium-dependent cell adhesion molecule that is expressed in a characteristic place-dependent pattern during development. Previous studies of ectopic expression of the chicken L-CAM gene under the control of heterologous promoters in transgenic mice suggested that cis-acting sequences controlling the spatiotemporal expression patterns of L-CAM were present within the gene itself. We have now examined the L-CAM gene for sequences that control its expression and have found an enhancer within the second intron of the gene. A 2.5-kb Kpn I-EcoRI fragment from the intron acted as an enhancer of a simian virus 40 minimal promoter driving a chloramphenicol acetyltransferase (CAT) reporter gene and produced 14.0-fold induction of CAT activity in MDCK cells. To narrow down the region responsible for enhancer activity and to determine whether the enhancer could function in a cell type-specific manner, a number of smaller restriction fragments from the intron were tested for activity in two chicken cell lines, the LMH hepatoma line, which produces high levels of L-CAM, and the SL-29 fibroblast line, which produces little, if any, L-CAM. Four L-CAM enhancer plasmids containing shorter segments derived from the intron showed enhanced CAT activity levels (between 9.4- and 16.5-fold) in extracts from transfected LMH cells but not from SL-29 cells. DNA sequence analysis of the L-CAM enhancer region revealed putative binding sites for the transcription factors SP1, E2A, and AP-2. In addition, LE-9, the smallest L-CAM enhancer segment (310 bp), contained a consensus binding site for the liver-enriched POU-homeodomain transcription factor, HNF-1. Tests of upstream sequences showed that a 630-bp fragment, corresponding to nearly the entire intergenic region between L-CAM and its neighboring CAM gene, K-CAM, could function as a promoter. In combination with the L-CAM enhancer, this fragment directed cell type-specific expression of the CAT reporter gene in LMH cells at a level comparable to that observed with enhancer constructs using the simian virus 40 minimal promoter. These combined observations define a promoter and an enhancer for the chicken L-CAM gene. They raise the possibility that these cis-acting regulatory sequences may be instrumental in directing specific place-dependent expression of the L-CAM gene in the chicken.