Pyrocatechol violet inhibits the initiation of protein synthesis in reticulocyte lysates. It prevents the formation of 80-S initiation complexes by inhibiting the binding of mRNA to 40-S initiation complexes. 2. Pyrocatechol violet inhibits the hydrolysis of methionyl-tRNAmetf by reticulocyte lysates or reticulocyte cell sap. 3. Concentrations of pyrocatechol violet sufficient to inhibit initiation have little effect on amino acid activation. Higher concentrations of pyrocatechol violet were strongly inhibitory.