Mammalian-secreted phospholipases A₂ (sPLA₂) form a diverse family of at least nine enzymes that hydrolyze phospholipids to release free fatty acids and lysophospholipids. We report here the cloning and characterization of human group IIF sPLA₂ (hGIIF sPLA₂). The full-length cDNA codes for a signal peptide of 20 amino acid followed by a mature protein of 148 amino acids containing all of the structural features of catalytically active group II sPLA₂s. hGIIF sPLA₂ gene is located on chromosome 1 and lies within a sPLA₂ gene cluster of about 300 kbp that also contains the genes for group IIA, IIC, IID, IIE, and V sPLA₂s. In adult tissues, hGIIF is highly expressed in placenta, testis, thymus, liver, and kidney. Finally, recombinant expression of hGIIF sPLA₂ in Escherichia coli shows that the enzyme is Ca²⁺-dependent, maximally active at pH 7–8, and hydrolyzes phosphatidylglycerol versus phosphatidylcholine with a 15-fold preference.