Cytokines are crucial in host defence against pathogens such as bacteria, viruses, fungi and parasites. A newly described cytokine, interleukin‐32 (IL‐32), induces various proinflammatory cytokines (tumour necrosis factor‐α, IL‐1β, IL‐6) and chemokines in both human and mouse cells through the nuclear factor‐κB and p38 mitogen‐activated protein kinase inflammatory signal pathway. The IL‐32 primarily acts on monocytic cells rather than T cells. In an attempt to isolate the IL‐32 soluble receptor, we used an IL‐32 ligand‐affinity column to purify neutrophil proteinase 3, which is a serine proteinase involved in many inflammatory diseases. IL‐32 has biological activity associated with Mycobacterium tuberculosis and chronic proinflammatory diseases such as rheumatoid arthritis. IL‐32 is transcribed as six alternative splice variants and the biological activity of each individual isoform remains unknown. Here, we cloned the complementary DNA of the four IL‐32 isoforms (α, β, γ and δ) that are the most representative IL‐32 transcripts. To produce recombinant protein with a high yield, the amino acids of two cysteine residues were mutated to serine residues, because serine residues are not conserved among different species. The multi‐step purified recombinant IL‐32 isoform proteins were assessed for their biological activities with different cytokine assays. The γ isoform of IL‐32 was the most active, although all isoforms were biologically active. The present study will provide a specific target to neutralize endogenous IL‐32, which may contribute to basic and clinical immunology.