Sphingosine-1-phosphate (S1P) induces a transient bradycardia in mammalian hearts through activation of an inwardly rectifying K⁺ current (I_KACh) in the atrium that shortens action potential duration (APD). We investigated probable mechanisms and receptor-subtype specificity for S1P-induced negative inotropy in isolated adult mouse ventricular myocytes. Activation of S1P receptors by S1P (100 nM) reduced cell shortening by ~25% (vs. untreated controls) in field-stimulated myocytes. S1P₁ was shown to be involved using the S1P₁-selective agonist SEW2871 on myocytes isolated from S1P₃-null mice. However, in these myocytes, S1P₃ can modulate a somewhat similar negative inotropy, as judged by the effects of the S1P₁ antagonist VPC23019. Since S1P₁ activates G_i exclusively, whereas S1P₃ activates both G_i and G_q, these results strongly implicate the involvement of mainly G_i. Additional experiments using the I_KACh blocker tertiapin demonstrated that I_KACh can contribute to the negative inotropy following S1P activation of S1P₁ (perhaps through G_i subunits). Mathematical modeling of the effects of S1P on APD in the mouse ventricle suggests that shortening of APD (e.g. as induced by I_KACh) can reduce I_CaL and decrease the [Ca⁺⁺]_i transient, both of which contribute to the observed negative inotropic effects of S1P. In summary, these findings suggest that the negative inotropy observed in S1P-treated adult mouse ventricular myocytes may consist of two distinctive components: (i) one pathway that acts via G_i to reduce I_CaL, blunt calcium-induced calcium release, and decrease [Ca⁺⁺]_i, and (ii) a second pathway that acts via G_i to activate I_KACh and reduce APD. This decrease in APD is expected to decrease Ca⁺⁺ influx and reduce [Ca⁺⁺]_i and myocyte contractility.