Intramembrane proteases regulate diverse processes by cleaving substrates within a transmembrane segment or near the membrane surface. Bacillus subtilis SpoIVFB is an intramembrane metalloprotease that cleaves Pro-σ(K) during sporulation. To elucidate features of Pro-σ(K) important for cleavage by SpoIVFB, coexpression of the two proteins in Escherichia coli was used along with cell fractionation. In the absence of SpoIVFB, a portion of the Pro-σ(K) was peripherally membrane associated. This portion was not observed in the presence of SpoIVFB, suggesting that it serves as the substrate. Deletion of Pro-σ(K) residues 2 to 8, addition of residues at its N terminus, or certain single-residue substitutions near the cleavage site impaired cleavage. Certain multiresidue substitutions near the cleavage site changed the position of cleavage, revealing preferences for a small residue preceding the cleavage site N-terminally (i.e., at the P1 position) and a hydrophobic residue at the second position following the cleavage site C-terminally (i.e., P2'). These features appear to be conserved among Pro-σ(K) orthologs. SpoIVFB did not tolerate an aromatic residue at P1 or P2' of Pro-σ(K). A Lys residue at P3' of Pro-σ(K) could not be replaced with Ala unless a Lys was provided farther C-terminally (e.g., at P9'). α-Helix-destabilizing residues near the cleavage site were not crucial for SpoIVFB to cleave Pro-σ(K). The preferences and tolerances of SpoIVFB are somewhat different from those of other intramembrane metalloproteases, perhaps reflecting differences in the interaction of the substrate with the membrane and the enzyme.