The interaction of cardiac fibroblasts (FBs) and myocytes (CMs) is gaining considerable interest due to growing evidence that fibroblasts can affect myocyte structure and function. This interaction can occur through direct cell contact or paracrine communication. Very limited data exists regarding paracrine communication, particularly between adult cells. We examined the effect of fibroblasts from normal and overloaded hearts on adult myocytes. TGF-β is a potential paracrine mediator and was investigated. Adult Lewis rat CMs were co-cultured with FBs from pressure overloaded (10 week thoracic aortic constriction (TAC)) (T+CM) or sham operated hearts (S+CM) at 1:2 CM/FB ratio in a co-culture system that allows paracrine communication but prevents direct cell contact. CMs co-cultured with CMs (CM+CM) served as control. After 24 hours co-culture, CM viability was measured as the % rod shaped cells. CM volume was measured using di-8-ANEPPS membrane staining. Ca2+ transients were recorded using the Ca2+ sensitive dye Fluo-4. 10 µM SB431542 (SB) was used to block TGF- β type 1 receptors. Data is shown as mean±SEM (n number) and analysed by 1 way ANOVA with Tukeys post hoc analysis. Co-culture with sham or TAC FBs reduced the viability of co-cultured CMs but this was blocked by SB (in %: CM+CM 70±1.1 (5); CM+CM+SB 72±1.7 (5); S+CM 61±2.3 (6); S+CM+SB 74±2.4 (6); T+CM 52±2.2 (6); T+CM+SB 66±2.8 (6). p<0.05: S+CM vs CM+CM, T+CM vs CM+CM, S+CM+SB vs S+CM, T+CM+SB vs T+CM). Both sham and TAC FBs induced CM hypertrophy which was also prevented by SB (in x103µm3: CM+CM 48±1.2 (60); CM+CM+SB 49±1.7 (43); S+CM 53±1.3 (48); S+CM+SB 48±1.4 (47); T+CM 56±1.7 (44); T+CM+SB 50±1.8 (42). p<0.05: S+CM vs CM+CM, T+CM vs CM+CM, S+CM+SB vs S+CM, T+CM+SB vs T+CM). Ca2+ transient amplitude was increased after co-culture with sham FBs but reduced after co-culture with TAC FBs. These effects were also prevented by SB (in F/F0: CM+CM 3.4±0.1 (42); CM+CM+SB 3.2±0.1 (33); S+CM 4.6±0.2 (50); S+CM+SB 3.3±0.1 (60); T+CM 2.8±0.1 (36); T+CM+SB 3.3±0.2 (45). p<0.05: S+CM vs CM+CM, T+CM vs CM+CM, S+CM+SB vs S+CM, T+CM+SB vs T+CM). Time to 90% decay of the Ca2+ transient was reduced after co-culture with sham or TAC FBs but unaffected by SB (in ms: CM+CM 359±8.5 (42); CM+CM+SB 390±17.2 (33); S+CM 322±6.2 (50); S+CM+SB 339±7.0 (60); T+CM 295±13.5 (36); T+CM+SB 317±9.3 (45). p<0.05: S+CM vs CM+CM, T+CM vs CM+CM). Time to peak and time to 50% decay were unaffected. These results show that FBs affect viability and Ca2+ cycling of CMs by paracrine TGF-β signalling. FBs from overloaded hearts alter Ca2+ cycling of CMs differently from normally loaded FBs, suggesting that this paracrine interaction is sensitive to chronic variations in load and may play a role in the pathophysiology of cardiac disease.