The transcription factor CREB (cAMP response element binding protein) mediates the mechanical strain-induced gene expression in the heart. We investigated which signaling pathways are involved in the CREB activation by strain using cultured ventricular fibroblasts from adult rat hearts. The analysis of phospho-CREB by immunocytochemistry showed a nearly complete CREB activation (93.2±6.1%) by cyclical mechanical strain (1 Hz, 5% elongation, 15 min) which did not distinguish from the whole number of CREB-positive fibroblasts (87.8±9.6%). The strain-induced CREB activation was partially reduced by PKA inhibition (19.9±11.2%), by PKC inhibition (31.2±11.2%) but not by CaMK II inhibition. The inhibition of several members of the MAPK cascade revealed a reduction of strain-induced CREB phosphorylation by 24.4±5.8% (Src-inhibition), by 27.7±5.8% (Raf1-inhibition) and by 23.8±8.6% (MEK-inhibition). The PI3-K inhibition reduced strain-caused phospho-CREB by 19.3±5.4%. The inhibition of p38, the stress-sensitive pathway, decreased the strain-induced CREB phosphorylation by 21.5±12.8%. Our results suggest that the complete strain-induced CREB phosphorylation includes several signaling pathways. We discuss this wide-ranging possibilities of CREB activation as safety for CREB-mediated gene expression in the heart.