Ubi-L, an isoform of the monoclonal nonspecific suppressor factor (MNSF), is an 8.5-kDa ubiquitin-like polypeptide. Ubi-L shows an antigen-nonspecific immunosuppressive action on various target cells including murine T helper type 2 clone, D10 cells. Most recently, we have characterized the biochemical nature of the receptor for Ubi-L. In this study, we observed that Ubi-L receptor ligation rapidly and transiently stimulated tyrosine phosphorylation of 65- and 31-kDa proteins in concanavalin A-activated D10 cells. The addition of neutralizing antibody to Ubi-L receptor inhibited the protein tyrosine phosphorylations and the Ubi-L-mediated suppression of IL-4 production by D10 cells. Genistein, a tyrosine kinase inhibitor, also reduced the induction of these protein tyrosine phosphorylations. IFN gamma, which is also known to inhibit the proliferative response of D10 cells, showed a synergistic effect with Ubi-L. Interestingly, IFN gamma enhanced the Ubi-L-induced tyrosine phosphorylation of the 31-kDa protein. These results suggest that tyrosine phosphorylation may be a key step in the initiation of the Ubi-L receptor-mediated transmembrane signaling,