Adult mouse ventricular myocytes (AMVMs) express S1P₁, S1P₂, and S1P₃ receptors. S1P activates Akt and ERK in AMVMs through a pertussis toxin-sensitive (G_i/o-mediated) pathway. Akt and ERK activation by S1P are reduced approximately 30% in S1P₃- and 60% in S1P₂- receptor knockout myocytes. With combined S1P_2,3 receptor deletion, activation of Akt is abolished and ERK activation is reduced by nearly 90%. Thus the S1P₁ receptor, while present in S1P_2,3 receptor knockout myocytes, is unable to mediate Akt or ERK activation. In contrast S1P induces pertussis toxin-sensitive inhibition of isoproterenol-stimulated cAMP accumulation in both WT and S1P_2,3 receptor knockout myocytes demonstrating that the S1P₁ receptor can functionally couple to G_i. An S1P₁ receptor selective agonist, SEW2871, also decreased cAMP accumulation but failed to activate ERK or Akt. To determine whether localization of the S1P₁ receptor mediates this signaling specificity, methyl--cyclodextrin (MCD) treatment was used to disrupt caveolae. The S1P₁ receptor was concentrated in caveolar fractions, and associated with caveolin-3 and this localization was disrupted by MCD. S1P-mediated activation of ERK or Akt was not diminshed but inhibition of cAMP accumulation by S1P and SEW2871 was abolished by MCD treatment. S1P inhibits the positive inotropic response to isoproterenol and this response is also mediated through the S1P₁ receptor and lost following caveolar disruption. Thus localization of S1P₁ receptors to caveolae is required for the ability of this receptor to inhibit adenylyl cyclase and contractility but compromises receptor coupling to Akt and ERK.