Liver cell adhesion molecule (L-CAM) is a calcium-dependent cell adhesion molecule found in very early vertebrate embryos and on liver and other epithelial cells in adults. To describe the genes coding for the molecule and study its synthesis, we have cloned cDNA from poly(A)+ RNA of 10-day embryonic chicken liver using the delta gt11 expression vector. One clone, lambda L301, has been characterized and used in analyses of L-CAM mRNA and genomic DNA. Clone lambda L301 produced a fusion protein that reacted strongly with polyclonal antibodies that recognize L-CAM (Mr 124,000) and its Mr 81,000 NH2-terminal fragment, Ft1, released from liver membranes by trypsin. This result indicates that lambda L301 contains a cDNA insert complementary to protein coding sequence within the two-thirds of the mRNA coding region beginning at the 5' end. The 220-base-pair cDNA insert was isolated and used as a probe in hybridization experiments. RNA transfer blot analysis of poly(A)+ RNA showed a single 4-kilobase mRNA; Southern blot analysis showed multiple components consistent with the presence of one to three L-CAM genes. To test whether different tissues express different forms of L-CAM message, poly(A)+ RNA from eight embryonic organs was analyzed. Only organs that expressed L-CAM protein contained poly(A)+ RNA that hybridized to the lambda L301 probe; in all cases a single band, with the same mobility as that in liver, was observed. The L-CAM mRNA in each tissue was present in proportions similar to those detected previously for the L-CAM protein in these tissues. The combined results suggest that any possible heterogeneity in the L-CAM genes is not reflected in the size of either the mRNA or protein.