Hematopoietic cell kinase (Hck) is a member of the Src family of non-receptor protein-tyrosine kinases that is expressed strongly in macrophages, an important target cell type for human immunodeficiency virus (HIV). The HIV Nef protein, a critical AIDS progression factor, binds Hck with unusually high affinity and induces Hck activation. Nef-mediated Hck activation has been proposed to trigger signaling pathways important for the establishment of HIV infection and subsequent progression to AIDS. Previous studies have utilized sequence alignments and binding assays to predict the functionality of Hck-Nef interactions, but these studies do not directly investigate the ability of Nef alleles to activate Hck. To better address this issue, we have created a model system in Saccharomyces cerevisiae based on previous findings that ectopic expression of c-Src arrests yeast growth in a kinase-dependent fashion. To establish the utility of this system for Hck and Nef, we first created a Hck variant containing a C-terminal sequence modified to encode the high-affinity SH2-domain binding sequence Tyr-Glu-Glu-Ile (HckYEEI). This modified sequence allows for autophosphorylation of the new tail and the subsequent intramolecular binding to the Hck SH2 domain. The resulting HckYEEI molecule models the physiologically down-regulated (inactive) form of Hck in structure and function. Unlike wild-type Hck, HckYEEI showed little kinase activity and failed to induce growth suppression in yeast. Importantly, co-expression with the Nef-SF2 allele activated HckYEEI, resulting in growth arrest. This effect was dependent on the conserved Nef proline-rich motif essential for Hck binding through its SH3 domain. However, Nef-SF2 did not activate FynYEEI, consistent with our previous data that the Fyn tyrosine kinase is not activated by Nef in mammalian cells. We then tested several additional laboratory HIV-1 Nef alleles for their ability to activate HckYEEI. Consensus, Lai, NL4.3, SF2, and YU-2 Nef alleles activated HckYEEI to varying degrees, as reflected by yeast growth inhibition. In contrast, Nef-Eli failed to suppress growth in the presence of HckYEEI, consistent with its inability to bind the Hck SH3 domain in vitro. Growth inhibition paralleled tyrosine phosphorylation of yeast proteins on anti-phosphotyrosine immunoblots. The differential effects of these Nef alleles on Hck activation correlated closely with previous results in mammalian cells. Taken together, these studies validate the use of yeast to reconstitute Nef-Hck interactions in a system amenable to screens of clinical Nef isolates, which may have predictive value in terms of AIDS progression.