17β-estradiol has been shown to protect both liver [1] and isolated hepatocytes [2] under pathophysiological conditions that have been associated with a rise in intracellular Ca²⁺ concentration. The classical mode of action of 17β-estradiol involves its translocation to the nucleus and regulation of transcription. However, numerous rapid non-genomic actions of 17β-estradiol have been demonstrated in several cell types, including stimulation of plasma membrane Ca²⁺-ATPase activity [3] and elevation of cGMP [4]. We have previously shown that another circulating hormone, atrial natriuretic peptide (ANP), stimulates hepatocyte plasma membrane Ca²⁺ efflux through elevation of cGMP and activation of protein kinase G (PKG), and have proposed that it may thus protect against Ca²⁺-dependent injury [5]. The aim of this study was to investigate the effects of 17β-estradiol on cGMP levels and plasma membrane Ca²⁺ efflux from rat hepatocytes. Hepatocytes were isolated from 150-250g male Wistar rats by collagenase digestion. cGMP was measured by enzymeimmunoassay. Ca²⁺ efflux from hepatocytes in suspension was measured spectrophotometrically, using membrane-impermeant fura-2 as extracellular Ca²⁺ reporter. Data are presented as mean ± SEM and are from at least 5 independent hepatocyte preparations. Significance of differences between means was assessed by Student’s t test (*significantly different from control at P < 0.05). We show that 10 nM 17β-estradiol rapidly elevates hepatocyte cGMP, which reaches peak values 5 min following addition of 17β-estradiol (266.2 ± 24.6* fmol/10⁶ cells, n = 12, compared with 183.8 ± 13.7 fmol/10⁶ cells, n = 7, in control, untreated hepatocytes). An optical isomer, 17α-estradiol, did not elevate cGMP (n = 11). However, the stimulatory effects of 17β-estradiol on cGMP production were mimicked by 17β-estradiol rendered membrane-impermeant by linkage to BSA (E2-BSA; n = 10). A basal rate of Ca²⁺ efflux was recorded from hepatocytes (99.9 ± 3.4 pmol/min/10⁶ cells, n = 117). Addition of 10 nM 17β-estradiol rapidly stimulated an approximate 1.5 fold increase in Ca²⁺ efflux (150 ± 9.6* pmol/min/10⁶ cells, n = 34). The stimulation of Ca²⁺ efflux was found to be dependent on the activation of PKG; in the presence of the PKG inhibitor, Rp-8-pCPT-cGMPS, there was no stimulation of Ca²⁺-efflux upon addition of 10 nM 17β-estradiol (n = 9). 17α-estradiol did not stimulate Ca²⁺ efflux (n = 10). However, the stimulatory effects of 17β-estradiol on Ca²⁺ efflux were mimicked by E2-BSA (n = 28). Taken together, these data suggest that 17β-estradiol has a rapid and specific effect on hepatocytes, acting at the plasma membrane to stimulate Ca²⁺ efflux through the generation of cGMP and activation of PKG.