Phosphorylation participates in the signaling, localization, and trafficking of G protein-coupled receptors. In this study, the amino acids known to be phosphorylated in α1A-adrenergic receptors were substituted with nonphosphorylatable residues in intracellular loop 3 (IL3) mutant, the carboxyl terminus (CTerm) mutant, and both structural regions (IL3-CTerm mutant). Their functions were compared with those of the wild-type (WT) receptor. Agonist- and protein kinase C-induced receptor phosphorylation was markedly decreased in the IL3-CTerm mutant, confirming the major roles of both domains in this process. Surprisingly, the substitutions did not affect the agonist-induced increase in intracellular calcium. Similarly, neither the WT nor the mutant receptors were desensitized by protein kinase C activation; however, agonist stimulation markedly desensitized the receptors, and this effect was diminished in the IL3 mutant, suggesting a role for this domain in this process. Extracellular signal-regulated kinase 1/2 phosphorylation was strong but transient in cells expressing the WT receptor, strong and sustained in those expressing the CTerm mutant, and markedly reduced in those expressing the IL3 and IL3-CTerm mutants. These findings suggest that IL3 phosphorylation sites modulate extracellular signal-regulated kinase activity. A weak and slow agonist-induced receptor-β-arrestin interaction was detected in cells expressing the WT receptor but was barely detectable in any of the mutants. Consistent with these findings, agonist- and phorbol ester-induced receptor internalization was observed in cells expressing the WT receptor but was markedly decreased in cells expressing the distinct mutants. SIGNIFICANCE STATEMENT: Phosphorylation sites in both intracellular loop 3 (IL3) and carboxyl terminus domains affect receptor function in distinct but complementary ways. The IL3 mutant showed decreased agonist-induced desensitization of the calcium response and agonist-activated extracellular signal-regulated kinase phosphorylation. α1A-Adrenergic receptor internalization and interaction with β-arrestin were decreased in all mutants containing phosphorylation site substitutions. Phosphorylation sites in the IL3 and carboxyl terminus domains appear to be functionally relevant for this receptor. The approach employed could be used to expand knowledge on other G protein-coupled receptors.