Ribosomes from a cryptopleurine-resistant mutant of yeast were analyzed to determine the subunit localization of the resistance alteration. As was previously reported (Grant, P., Sanchez, L., and Jiminez, A. (1974), J. Bacteriol. 120, 1308), in vitro resistance of polyphenylalanine synthesis to cryptopleurine was conferred by 40S subunits from the mutant. Binding studies with sensitive ribosomes were carried out in order to identify the subunit binding site for cryptopleurine. Over the range of concentrations which inhibited polyphenylalanine synthesis, binding was proportional to concentration, so that a unique binding site could not be detected. Furthermore, binding to isolated subunits was about fourfold greater than to 80S ribosomes, suggesting that non-specific binding was sensitive to the condition of the particles. Model systems were developed in order to determine which step of the elongation cycle was inhibited by cryptopleurine. Elongation factor 1 dependent binding of Phe-tRNA to ribosomes was not inhibited by cryptopleurine concentrations, which inhibited polyphenylalanine synthesis. The initial rate of N-acetylphenylalanylpuromycin formation was inhibited when 10(-5) M cryptopleurine was added prior to translocation, but not when added after. Little inhibition was observed in either case when mutant ribosomes were used. These results suggest that cryptopleurine primarily inhibited translocation.