'Regulators of G protein signaling' (RGS proteins) are members of a large family of GTPase-activating proteins that are differentially expressed in various cell types and accelerate the termination of heterotrimeric G protein signaling. To identify RGS proteins that may affect autonomic regulation of atrial excitability, we screened the expression of nineteen RGS genes (RGS subfamilies A, B, C, and D) in single spontaneously beating rat atrial myocytes maintained in primary culture. Expression profiling by single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that seven distinct RGS genes are endogenously expressed in atrial myocytes which were also identified in poly(A)(+) mRNA from rat atria (RGS2, RGS3, RGS4, RGS6, RGS10, GAIP, and RGSZ2). Other RGS transcripts were detected in atrial poly(A)(+) mRNA but not single atrial myocytes (RGS5, RGS12, RGS16, and RGS18), and therefore are likely to originate from non-myocyte sources in atrial tissue. The single-cell RT-PCR experiments also led to the identification of putative splice variants for RGS6 and GAIP. Immunocytochemistry using RGS-specific antibodies confirmed the presence of selected RGS proteins in the cultured atrial myocytes. These results demonstrate a rich diversity of RGS expression in atrial myocytes whose specific role in G-protein signaling is yet to be determined. The identification of endogenous RGS proteins in atrial myocytes will facilitate targeted suppression and/or deletion studies to determine how each RGS protein may affect atrial excitability and its short-term and long-term regulation by G-protein signaling events.