Mammalian defensins are cationic antimicrobial peptides that play a central role in host innate immunity and as regulators of acquired immunity. In animals, three structural defensin subfamilies, designated as alpha, beta, and , have been characterized, each possessing a distinctive tridisulfide motif. Mature alpha- and beta-defensins are produced by simple proteolytic processing of their prepropeptide precursors. In contrast, the macrocyclic -defensins are formed by the head-to-tail splicing of nonapeptides excised from a pair of prepropeptide precursors. Thus, elucidation of the -defensin biosynthetic pathway provides an opportunity to identify novel factors involved in this unique process. We incorporated the -defensin precursor, proRTD1a, into a bait construct for a yeast two-hybrid screen that identified rhesus macaque stromal cell-derived factor 2-like protein 1 (SDF2L1), as an interactor. SDF2L1 is a component of the endoplasmic reticulum (ER) chaperone complex, which we found to also interact with alpha- and beta-defensins. However, analysis of the SDF2L1 domain requirements for binding of representative alpha-, beta-, and -defensins revealed that alpha- and beta-defensins bind SDF2L1 similarly, but differently from the interactions that mediate binding of SDF2L1 to pro--defensins. Thus, SDF2L1 is a factor involved in processing and/or sorting of all three defensin subfamilies.