Human mesenchymal stem cells (hMSCs) are a multipotent cell population with the potential to be a cellular repair or delivery system provided that they communicate with target cells such as cardiac myocytes via gap junctions. Immunostaining revealed typical punctate staining for Cx43 and Cx40 consistent with along regions of intimate cell-to-cell contact between hMSCs. The staining patterns for Cx45 rather were typified by granular cytoplasmic staining. hMSCs exhibited cell-to-cell coupling to each other, to HeLa cells transfected with Cx40, Cx43, Cx45 and to acutely isolated canine ventricular myocytes. The junctional currents (Ij) recorded between hMSC pairs exhibited quasi-symmetrical and asymmetrical voltage (Vj) dependence. Ij records from hMSC-Cx43HeLa and hMSC-Cx40HeLa cell pairs also showed symmetrical and asymmetrical Vj-dependence, while hMSC-Cx45HeLa pairs always produced asymmetric Ij with pronounced Vj-gating when the Cx45 side was negative. Symmetric Ij suggests that the dominant functional channel is homotypic, while the asymmetric Ij suggests the activity of another channel type (heterotypic, heteromeric or both). The hMSCs exhibited a spectrum of single channels with transition conductances (j) of 30-80 pS. The macroscopic Ij obtained from hMSC-cardiac myocyte cell pairs exhibited asymmetrical Vj-dependence, while single channel events revealed j of the size range 40-100 pS. hMSCs coupling via gap junctions to other cell types provides the basis for considering them as a therapeutic repair or cellular delivery system to syncytia such as the myocardium.