Embryos of the brine shrimp, Artemia salina, were used in a study of polypeptide chain initiation in an in vitro system from a eukaryote. A protein, isolated from the high-speed supernatant, has been highly purified and shown to have properties that suggest it is the eukaryotic equivalent of the Escherichia coli initiation factor F(2): It promotes the AUG-dependent binding of fMet-tRNA (E. coli) to the Artemia 40S ribosomal subunit, but not to either the 60S or 80S species; the bound fMet-tRNA is placed in a site on the smaller subunit from which it reacts with puromycin upon addition of the 60S subunit; and the activity is sensitive to aurintricarboxylic acid and edeine, specific inhibitors of initiation. The factor, a basic protein of molecular weight about 100,000, is inactivated by N-ethylmaleimide, an SH-binding reagent, and is clearly distinct from the Artemia elongation factors, T(1) and T(2). In addition, the factor stimulates the poly(U)-dependent binding of Phe-tRNA (E. coli) to the Artemia 40S ribosomal subunit. This reaction, though similar to the fMet-tRNA-binding reaction, differs in that the bound Phe-tRNA is largely resistant to release by puromycin.