The presence of a basal nonselective cation permeability was mainly investigated in primary cultures of rat cardiac microvascular endothelial cells (CMEC) by applying both the patch-clamp technique and Fura-2 microfluorimetry. With low EGTA in the pipette solution, the resting membrane potential of CMEC was -21.2 +/- 1.1 mV, and a Ca(2+)-activated Cl(-) conductance was present. When the intracellular Ca(2+) was buffered with high EGTA, the membrane potential decreased to 5.5 +/- 1.2 mV. In this condition, full or partial substitution of external Na(+) by NMDG(+) proportionally reduced the inward component of the basal I-V relationship. This current was dependent on extracellular monovalent cations with a permeability sequence of K(+) > Cs(+) > Na(+) > Li(+) and was inhibited by Ca(2+), La(3+), Gd(3+), and amiloride. The K(+)/Na(+) permeability ratio, determined using the Goldman-Hodgkin-Katz equation, was 2.01. The outward component of the basal I-V relationship was reduced when intracellular K(+) was replaced by NMDG(+), but was not sensitive to substitution by Cs(+). Finally, microfluorimetric experiments indicated the existence of a basal Ca(2+) entry pathway, inhibited by La(3+) and Gd(3+). The basal nonselective cation permeability in CMEC could be involved both in the control of myocardial ionic homeostasis, according to the model of the blood-heart barrier, and in the modulation of Ca(2+)-dependent processes.