The Na,K-ATPase provides the driving force for many ion transport processes through control of Na+ and K+ concentration gradients across the plasma membranes of animal cells. It is composed of two subunits, cu and beta, In many tissues, predominantly in kidney, it is associated with a small ancillary component, the gamma -subunit that plays a modulatory role. A novel 15-kDa protein, sharing considerable homology to the gamma -subunit and to phospholemman (PLM) was identified in purified Na,K-ATPase preparations from rectal glands of the shark Squalus acanthias, but was absent in pig kidney preparations. This PLM-like protein from shark (PLMS) was found to be a substrate for both PKA and PKC, Antibodies to the Na,K-ATPase or subunit coimmunoprecipitated PLMS, Purified PLMS also coimmunoprecipitated with the ol-subunit of pig kidney Na,K-ATPase, indicating specific association with different alpha -isoforms. Finally, PLMS and the alpha -subunit were expressed in stoichiometric amounts in rectal gland membrane preparations. Incubation of membrane bound Na,K-ATPase with non-solubilizing concentrations of C12E8 resulted in functional dissociation of PLMS from Na,K-ATPase and increased the hydrolytic activity. The same effects were observed after PKC phosphorylation of Na,K-ATPase membrane preparations. Thus, PLMS may function as a modulator of shark Na,K-ATPase in a way resembling the phospholamban regulation of the Ca-ATPase.