Intracellular calcium (Ca²⁺) plays pivotal roles in distinct cellular functions through global and local signaling in various subcellular compartments; and subcellular Ca²⁺ signal is the key factor for independent regulation of different cellular functions. In vascular smooth muscle cells, sub-sarcolemmal Ca²⁺ is an important regulator of excitation-contraction coupling, and nucleoplasmic Ca²⁺ is crucial for excitation-transcription coupling. However, information on Ca²⁺ signals in these subcellular compartments is limited. To study the regulation of the subcellular Ca²⁺ signals, genetically encoded Ca²⁺ indicators (cameleon), D3cpv, targeting the plasma membrane (PM), cytoplasm and nucleoplasm were transfected into rat pulmonary arterial smooth muscle cells (PASMCs) and Ca²⁺ signals were monitored using laser scanning confocal microscopy. In situ calibration showed that the K_d for Ca²⁺ of D3cpv was comparable in the cytoplasm and nucleoplasm, but it was slightly higher in the PM. Stimulation of digitonin permeabilized cells with IP3 elicited a transient elevation of [Ca²⁺] with similar amplitude and kinetics in the nucleoplasm and cytoplasm. Activation of G-protein coupled receptors by endothelin-1 and angiotensin-II preferentially elevated the subsarcolemmal Ca²⁺ signal with higher amplitude in the PM region than the nucleoplasm and cytoplasm. In contrast, the receptor tyrosine kinase activator, platelet derived growth factor, elicited Ca²⁺ signals with similar amplitudes in all three regions, except the rise-time and decay-time were slightly slower in the PM region. These data clearly revealed compartmentalization of Ca²⁺ signals in the subsacrolemmal regions and provide the basis for further investigations of differential regulation of subcellular Ca²⁺ signals in PASMCs.