The spontaneous dissociation of complexes containing elongation factor G (EF-G), the ribosome, and either GDP plus fusidic acid, guanyl-5'-yl imidodiphosphate, or guanyl-5'-yl methylene diphosphonate has been measured and it follows biphasic kinetics that can be resolved into two first-order decay rates. This suggest the existence of two classes of complexes with apparent dissociation rate constants (k) differing 5--20-fold. The values of k and the distribution of complexes between the fast and the slowly decaying class depend on the conditions in which the dissociation occurs but not on the conditions in which the complexes are formed. Rapid transitions of complexes from one to the other class occur only when the chemical environment in which the dissociation takes place is modified. Thus, increasing the concentration of NH4Cl or adding the antibiotic thiostrepton accelerates the decay and converts slowly dissociating into fast dissociating complexes. In contrast, addition of misreading-inducing aminoglycoside antibiotics of the neomycin, kanamycin, streptomycin, and gentamicin (but not hygromycin) groups slows down the decay. For neomycin B at 10 micron, this effect is due to the conversion of fast into slowly decaying complexes. A model to explain the results involving conformational transitions of the complexes is proposed.