Many membrane proteins are implicated in the control of cell function by triggering specific signaling pathways. There is a new family of membrane proteins, defined by its structural motifs, which includes several lymphoid antigens, but lacks a function. To study its biological role, we determined which signaling pathways are affected by the CD53 antigen, a prototypic member of this family, in rat macrophages. Activation of CD53 by cross-linking results in an increase in inositol phosphates and diacylglycerol and in Ca2+ mobilization, which are insensitive to pertussis or cholera toxins. There is a translocation of protein kinase C to the membrane accompanied by nitric oxide (NO) release in macrophages. This effect is the result of the expression of the inducible nitric oxide synthase (iNOS), which is dependent on protein kinase C and protein synthesis. These results have linked a new receptor with a specific pathway of NO induction and thus have opened up a novel aspect of NO regulation in cell biology.