The nuclear import of 5-lipoxygenase modulates its capacity to produce leukotrienes from arachidonic acid. However, the molecular determinants of its nuclear import are unknown. Recently, we used structural and functional criteria to identify a novel import sequence at Arg(518) on human 5-lipoxygenase (Jones, S. M., Luo, M., Healy, A. M., Peters-Golden, M., and Brock, T. G. (2002) J. Biol. Chem. 277, 38550-38556). However, this analysis also indicated that other import sequences must exist. Here, we identify two additional sites, at Arg(112) and Lys(158), as nuclear import sequences. Both sites were found to be common to 5-lipoxygenases from different species but not found on other lipoxygenases. Both sites also appeared to be a part of structures that were predominantly random loops. Peptide sequences at these sites were sufficient to direct nuclear import of green fluorescent protein. Mutation of basic residues in these sites impaired nuclear import and combinations of mutations at different sites were additive in effect. Mutations in all three sites were required to disable nuclear accumulation of 5-lipoxygenase in all cells. Significantly, mutation in these sites did not inhibit catalytic function. Taken together, these results indicate that nuclear import of 5-lipoxygenase may reflect the combined functional effects of three discrete import sequences. Mutation of individual sites can, by itself, impair nuclear import, which in turn could impact arachidonic acid metabolism.