The human cell surface CD38 molecule is a 46-kDa type-II transmembrane glycoprotein with a short N-terminal cytoplasmic domain and a long Cys-rich C-terminal extracellular one. We previously demonstrated that an ecto-form NAD+ glycohydrolase (NADase) activity induced by all-trans retinoic acid in HL-60 cells is due to the extracellular domain of CD38. In the present study, we investigated a possible signal transduction mediated through CD38 in the retinoic acid-differentiated HL-60 cells with anti-CD38 monoclonal antibodies (mAbs). The addition of selected anti-CD38 mAbs to the cells induced rapid tyrosine phosphorylation of the cellular proteins with the molecular weights of 120,000, 87,000 and 77,000; the phosphorylated 120-kDa protein was identified as the c-cbl proto-oncogene product, p120c-cbl. Furthermore, the phosphorylated p 120c-cbl associated with the 85-kDa subunit of phosphatidylinositol 3-kinase. To determine the relationship between the amino acid sequence responsible for the NADase activity and epitopes recognized by the stimulatory mAbs, we produced its carboxy-terminal deletion mutants in COS-7 cells. The mutants with less than 15 amino acids deleted from the carboxyl terminus of the 300-amino acid wild-type molecule still maintained NADase activity, but those with more than 27 amino acids deleted did not. Introduction of site-directed mutation of a cysteine residue (Cys275), located in the 273-285 sequence, completely abolished the NADase activity. These CD38 mutants were also used for an epitope mapping of anti-CD38 mAbs. All the epitopes recognized by the mAbs inducing the tyrosine phosphorylation were mapped on the same Cys275-containing sequence of 273-285. Thus, the discrete carboxy-terminal sequence not only plays a key role in its ecto-NADase activity, but also contains the epitopes of the agonistic anti-CD38 mAbs for the transmembrane signaling. We also found that the agonistic mAbs markedly potentiate superoxide generation induced by the stimulation of G protein-coupled chemotactic receptors. Our results suggested that the stimulation of CD38 might generate an accessory signal(s) to enhance the G protein-mediated signaling, probably though the protein-tyrosine phosphorylation.