The effects of the Guanosine 5'-triphosphate analog guanylylimidodiphosphate and several antibiotics on protein synthesis in a cell-free system from rat brain were investigated as a function of the template used. Polypeptide chain elongation was promoted by the analog only in the system coded by the endogenous template but not when coded by polyuridylic acid. The effects of the antibiotics were also dependent on the template used. This suggest that the template conditioned the ribosomal conformation which determine the inhibitory effect of the antibiotics.