A lambda gt11 human hepatoma cDNA expression library was screened with antibodies to human alpha-L-fucosidase, a lysosomal enzyme whose activity is deficient in the human autosomal recessive disease fucosidosis. Three positive clones were identified after screening 9 X 10(6) plaques. One of these was sequenced and found to be spurious, probably representing an out-of-frame cDNA that gave rise to amino acid sequences of unknown length that crossreacted with alpha-L-fucosidase. A second clone, lambda AF3, was isolated which, after establishment in Escherichia coli BNN103, gave rise to a fusion protein of Mr 154,000 containing a human fragment of Mr 40,000 that represented 80% of the mature processed enzyme (Mr 50,000). Southern blot analysis of mouse and human chromosomal DNA confirmed the human origin of insert AF3. The nucleotide sequence of AF3 was determined and colinearity was established between 270 nucleotides and 90 amino acids in alpha-L-fucosidase. AF3 was found to contain 1058 base pairs and to code for 347 amino acids of alpha-L-fucosidase. Four potential glycosylation sites were identified. The frequency of lambda AF3 in the hepatoma library was 0.0018%.