Human immunodeficiency virus Nef plays an important role in AIDS pathogenesis. In addition to the well known down-regulation of cell surface receptors (CD4, MHCI), Nef is able to alter cellular signaling. Of particular interest for this study is the ability of Nef to bind with a very high affinity to SH3 domains of myelomonocyte-specific protein-tyrosine kinases of the Src family (Src-like PTK). We have therefore investigated Ca(2+) signaling in HL60 cells retrovirally transduced with wild type Nef or with a Nef mutant deficient in the SH3-interacting proline-rich motif (Nef((PXXP)4(-))). In differentiated HL60 cells, Nef markedly altered cellular Ca(2+) signaling; the amount of intracellularly stored Ca(2+) was increased, and as a consequence, store-operated Ca(2+)-influx was decreased. This effect was not observed in undifferentiated HL60 cells or in CEM T-lymphocytes and correlated with the differentiation-induced up-regulation of Src-like PTK. The Nef effect on Ca(2+) signaling depended entirely on the integrity of its PXXP motif. The Src-like PTK p56/59(hck) co-immunoprecipitated with both Nef and with the inositol 1,4,5-trisphosphate receptor, providing a possible mechanistic link between the viral protein and intracellular Ca(2+) stores of the host cell. Collectively, our results demonstrate that the human immunodeficiency virus 1 Nef protein manipulates intracellular Ca(2+) stores through SH3-mediated interactions in myelomonocytic cells.