Guinea pig mesenteric lymphatic vessels exhibit rhythmic constrictions induced by action potential (AP)-like spikes and initiated by entrainment of spontaneous transient depolarizations (STDs). To characterize STDs and the signaling mechanisms responsible for their occurrence, we used intracellular microelectrodes, Ca²⁺ imaging and pharmacological agents. In our investigations on the role of intracellular Ca²⁺ released from Ca²⁺ stores, we observed that intracellular Ca²⁺ transients accompanied some STDs though there were many exceptions where Ca²⁺ transients occurred without accompanying STDs. STD frequency and amplitude were markedly affected by activators/inhibitors of inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃Rs), but not by treatments known to alter Ca²⁺ release via ryanodine receptors (RyRs). A role for Ca²⁺-activated chloride (Cl_Ca) channels was indicated, as STDs were dependent on the Cl^- but not Na+ concentration of the superfusing solution and were inhibited by Cl_Ca channel blockers niflumic acid (NFA), anthracene 9-carboxylic acid (9-AC) and NPPB but not by the Cl_vol blocker DIDS. Increases in STD frequency and amplitude induced by agonist stimulation were also inhibited by NFA. Nifedipine, the hyperpolarization-activated inward current blocker ZD7288, the non-selective cation/store-operated channel blockers SKF96365, Gd³⁺ and Ni²⁺ had no or marginal effects on STD activity. However, nifedipine, 2-APB, NFA, SKF96365, Gd³⁺ and Ni²⁺ altered the occurrence of spontaneous APs. Our findings support a role for Ca²⁺ release through IP3Rs and resultant opening of Cl_Ca channels in STD generation and confirm the importance of these events in the initiation of lymphatic spontaneous APs and subsequent contractions. Abolition of spontaneous APs by blockers of other excitatory ion channels suggests a contribution of these conductances to lymphatic pacemaking.