All primate lentiviruses encode Nef, an accessory protein that is important for viral pathogenicity in vivo. Lentiviral Nef proteins regulate the release of chemokines (MIP-1 alpha/beta) from infected macrophages, thereby enhancing virus dissemination (S. Swingler, A. Mann, J. Jacque, B. Brichacek, V. G. Sasseville, K. Williams, A. A. Lackner, E. N. Janoff, R. Wang, D. Fisher, and M. Stevenson, Nat. Med. 5:997-1003, 1999). In the current study, we have identified a novel domain within Nef (K(92)EK) that is required for Nef-dependent MIP-1beta production by infected macrophages. Mutations in this domain abrogated MIP-1beta induction but did not affect other Nef-ascribed activities, such as CD4 or major histocompatibility complex (MHC) class Iota downregulation. This further underscores Nef as a modular protein with genetically separable activities that may contribute to its role in viral replication and pathogenicity.