The objective of this work was to evaluate the effects of testosterone (T) and 17-estradiol (E₂) on coronary microvascular endothelial cells (CMEC) of male and female rats. To analyze the short-term effects of such sexual steroid hormones on intracellular calcium concentration ([Ca²⁺]_i) kinetics we used chelating agents (FURA 2 AM). We also explored the possibility of testosterone aromatization by using selective inhibitors of the aromatase enzyme (P450_AROM); aminoglutethimide (4µM and 4-hydroxyandrostenedione (4µM. The presence of P450_AROM was investigated by immunocytochemical and immunoblot assays, using peptide-generated polyclonal antibodies raised against a 20-amino acid synthetic fragment of the rat P450_AROM and by in situ hybridization to locate the aromatase RNAm in such cells. The activity of P450_AROM was demonstrated by the stereospecific loss of the tritium atom of [1-³H] androstenedione. Our results indicate that both T and E₂ induced a rapid increase in [Ca²⁺]_i. The fact that E₂ and T effects were carried out within milliseconds suggests that they were exerted at the membrane level and not through intracellular receptors. The possibility of involvement of the Phospholipase C (PLC) in these effects is suggested since U-73122, a PLC inhibitor, blocked both T and E₂ effects. Immunocytochemical assays indicated the expression of androgenic and estrogenic receptors in these cells. T effects were blocked by the selective aromatase inhibitors. We also demonstrated membrane-association of P450_AROM., the expression of the ovary-specific mRNA after in situ hybridization and E₂ formation resulting from a significant activity of P450_AROM in CMEC. There were not any gender-based differences.