Protein synthesis initiation factors present in a crude 0.5 M KCL microsomal wash fraction were isolated from the livers of immature rats that had been injected either 2 or 17 hours earlier with the plycyclic hydrocarbon, 3-methylcholanthrene (3MC), and then were tested for their ability to stimulate natural mRNA-directed protein synthesis in vitro. After purification of the initiation factors by means of ammonium sulfate fractionation, and DEAE-cellulose and Sephadex G-200 chromatography, M2A and M2B, but not M3 or M1, from the livers of 3MC-pretreated rats were more active than were similarly-prepared control factors in their ability to initiate the synthesis of rabbit globin polypeptides in a highly fractionated cell-free protein synthesizing system derived from rabbit reticulocytes. The greater activity of the M2A and M2B preparations from drug-treated rat liver did not appear to be due to differences in the composition of initiation factor protein extracted from the livers of control or experimental rats and occurred very early after administration of 3MC, i.e., 2 hr. The role of the protein synthesis initiation factors in the altered rates of protein synthesis which accompany cytodifferentiation and growth is discussed.