Gap junctions allow rapid exchange of ions and small metabolites between cells. They can occur between connective tissue cells, and in tendons there are two prominent types, composed of connexin 32 or 43. These form distinct networks - tenocyte rows are linked by both longitudinally, but only by connexin 43 laterally. We hypothesised that the junctions had different roles in cell response to mechanical loading, and measured the effects of inhibitors of gap junction function on secretion of collagen by tenocyte cultures exposed to mechanical strain. Chicken tendon fibroblasts were exposed to cyclic tensile loading in the presence or absence of general gap junction inhibitors (halothane or the biomimetic peptide gap27), or antisense oligonucleotides to chicken connexin 32 or 43. Untreated cultures increased collagen secretion by around 25% under load. Halothane eliminated this response but caused cell damage. Gap27 peptide reduced secretion but maintained loading effects - strained cultures secreting more collagen than unstrained. Antisense downregulation showed major differences between connexins: antisense 32 reduced, and antisense 43 increased, collagen secretion. In both cases loading effects were maintained. This shows that (i) gap junctional integration of signals is important in load response of tenocyte populations - mechanotransduction occurs in individual cells but integration of signals markedly enhances it and (ii) communication via connexin 32 and 43 have differential effects on the load response, with connexin 32 being stimulatory and connexin 43 being inhibitory. Cells coordinate and control their response to mechanical signals at least in part by differential actions of these two types of gap junction.