Abstract Recombinant human interleukin-5 (rhIL-5) was expressed in baculovirus-infected insect cells and purified to homogeneity from the culture medium in a single chromatographic step. Beginning with a cDNA encoding the full-length precursor form of human IL-5, including the authentic secretory leader sequence, recombinant baculovirus-infected insect cells expressed high levels of rhIL-5 (5-15 mg/liter culture) of which >90% was processed to the mature form and secreted into the culture medium. After removing cells by centrifugation, rhIL-5 was purified by first adjusting the culture medium to the calculated p I value of mature IL-5 (p I 7.44) and then passing the conditioned medium through tandem linked anion- and cation-exchange columns. The resulting pass-through fraction contained the rhIL-5 and was devoid of contaminating proteins. An optional hydrophobic-interaction chromatography step effectively concentrated the pure homodimeric N-glycosylated rhIL-5 with a high overall yield (>90%). N-terminal amino acid sequence determination indicated that cleavage of the human IL-5 leader sequence in insect cells occurred between Ala 19 and Ile 20. Recombinant human IL-5 prepared by this procedure bound to the high-affinity IL-5 receptor present on an eosinophilic leukemia cell line and elicited a proliferative response in the IL-B-dependent murine B-celI line BCL 1. This rapid and simple procedure for the expression and purification of mature rhIL-5 should therefore enable studies requiring large amounts of this cytokine.