Human serum alpha-L-fucosidase has been purified 241 200-fold with 35% yield by an affinity chromatographic procedure utilizing agarose-epsilon-aminocaproyl-fucosamine. Isoelectric focusing of the purified enzyme indicated the presence of several forms, with the form at pI 5.0 comprising the majority of the activity. Assay of the purified alpha-L-fucosidase showed only trace amounts of contaminating glycosidases present, with beta-galactosidase being the largest contamnant (0.5% by activity). Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated the presence of two subunits with very similar molecular weights (56 500 and 54 000). Using the p-nitrophenyl substrate, the purified serum alpha-L-fucosidase has an apparent Michaelis constant of 0.52 mM and a broad pH optimum centered around pH 4.8 with a second, minor optimum at pH 6.1. Gel filtration on Sepharose 6-B indicated an apparent molecular weight of 296 000 +/- 30 000. Preincubation with antibodies made previously against purified liver alpha-L-fucosidase led to quantitative immunoprecipitation of the purified serum alpha-L-fucosidase. Assay of the purified serum alpha-L-fucosidase for sialic acid indicated the presence of 1.7 microgram sialic acid per 100 microgram enzyme, about twice that previously found for the purified liver enzyme.