The lipoxygenases (LOs) are a family of nonheme iron dioxygenases that catalyse the insertion of molecular oxygen into polyunsaturated fatty acids. Five members of this gene family have been described in man, 5-LO, 12S-LO, 12R-LO, 15-LO and 15S-LO. Using partially purified recombinant 15S-LO enzyme and cells constitutively expressing this protein, we have compared the activity, substrate specificity, kinetic characteristics and regulation of this enzyme to that previously reported for 15-LO. 15S-LO has a threefold higher Km, similar Vmax and increased specificity of oxygenation for arachidonic acid, and a similar Km but decreased Vmax for linoleic acid in comparison to 15-LO. Unlike 15-LO, 15S-LO is not suicide inactivated by the products of fatty acid oxygenation. However, in common with other LOs, 15S-LO activity is regulated through calcium-dependent association of the enzyme with the membrane fraction of cells. In addition, whilst independently cloning the recently described 15S-LO, we identified a splice variant containing an in-frame 87-bp deletion corresponding to amino acids 401-429 inclusive. Modelling of the 15S-LO and subsequent studies with partially purified recombinant protein suggest that the deleted region comprises a complete alpha-helix flanking the active site of the enzyme resulting in decreased specificity of oxygenation and affinity for fatty acid substrates. Alternative splicing of 15S-LO would therefore provide a further level of regulation of fatty acid metabolism. These results demonstrate that there are substantial differences in the enzyme characteristics and regulation of the 15-LO isozymes which may reflect differing roles for the proteins in vivo.