Although they have distinct functions, the signaling of dopamine-D sub(2) receptor short and long isoforms (D sub(2)S and D sub(2)L) is virtually identical. We compared inhibitory regulation of extracellular signal-regulated kinases (ERK12) in GH4 pituitary cells separately transfected with these isoforms. Activation of rat or human dopamine-D sub(2)S, muscarinic or somatostatin receptors inhibited thyrotropin-releasing hormone-induced ERK12 phosphorylation, while the D sub(2)L receptor failed to inhibit this response. In order to address the structural basis for the differential signaling of D sub(2)S and D sub(2)L receptors, we examined the D sub(2)L-SS mutant, in which a protein kinase C (PKC) pseudosubstrate site that is present in the D sub(2)L but not D sub(2)S receptor was converted to a consensus PKC site. In transfected GH4 cells, the D sub(2)L-SS mutant inhibited thyrotropin-releasing hormone-induced ERK12 phosphorylation almost as strongly as the D sub(2)S receptor. A D sub(2)S-triple mutant that eliminates PKC sites involved in D sub(2)S receptor desensitization also inhibited ERK12 activation. Similarly, in striatal cultures, the D sub(2)-selective agonist quinpirole inhibited potassium-stimulated ERK12 phosphorylation, indicating the presence of this pathway in neurons. In conclusion, the D sub(2)S and D sub(2)L receptors differ in inhibitory signaling to ERK12 due to specific residues in the D sub(2)L receptor alternatively spliced domain, which may account for differences in their function in vivo.