Cardiac fibroblasts (CF) are emerging as key components of normal cardiac function, as well as the response to stressors and injury, by interacting with cardiomyocytes (CM) via paracrine mechanisms and direct cell-cell interactions (Chilton et al, 2007; Kakkar and Lee, 2010). We have previously shown that in a direct contact cell-to-cell or by the use of cell inserts, CF leads to reduced calcium contractility and prolonged time of calcium decay in sedentary rats. We aimed to test on CF and CM interaction, a program of exercise training (TR), which mimics some therapeutics agents, and has been shown to improve cardiomyocytes (CM) contractility (Wisloff et al, 2001). Adult Female Spague-Dawley rats, n=35, weighting 220-350g, were given inhalational anesthesia with isoflurane (5% for 2-3 min). CM and CF were isolated as previously described with minor modifications for CF centrifugation time (Guatimosim et al, 2002). We used a noncontact protocol by keeping freshly isolated cardiomyocytes in conditioned media from CF of trained (TR) and sedentary (SED) rats, CF were used when they achieved 60-70% of confluence. Epifluorescence measurements (using Fura 2) have shown reduced Ca2+ transient amplitude in cardiomyocytes (n>7 CM for each treatment) (Control: 0,198±0.02, vs SED FB: 0,118±0,007, vs TR FB: 0,095±0,008, fura ratio, p<0.05,) and reduced shortening (Control: 6,2±1.1, vs SED FB: 2,23±0.29, vs TR FB: 2.52±0,56, % of shortening, p<0.05), but not diastolic calcium removal as measured as time interval to 50% decay of Ca2+ concentration (Control: 0.176±0.01, vs SED FB: 0.222±0.01, vs TR FB: 0.223±0,03, seconds, p<0.05) when CM were alone (Control) or together with SED FB or TR FB, for 18h. Values are means ± S.E., compared by ANOVA. Conditioned media did not have altered pH compared to control media (approximately 7.67 and 7.68, pH, respectively). We initially conclude that CF from SED and TR rats have paracrine secretion which leads to reduced CM maximum rate of shortening and reduced Ca2+ transient amplitude and this does not seem to be related to altered media pH.