Extracts of folate-deficient Streptococcus faecium have been examined for their ability to dispense with the normal requirement of formylation of methionyl-tRNA to initiate polypeptide biosynthesis. [ 35S]Methionyl-tRNA's prepared from Escherichia coli or S. faecium both require formylation to bind to S. faecium ribosomes in the presence of endogenous mRNA and AUG. Formation of the puromycin derivative of the initiator by S. faecium components is similarly formylation dependent. Spermine promotes a further, but non-specific, Mg 2+-independent ribosomal binding of both methionyl-tRNA and formylmethionyl tRNA. The cell-free system differs from the intact cell which can utilize formylated or unformylated initiator, and from the eucaryote cytoplasm which does not formylate the initiator. A factor of the intact cell of S. faecium is postulated to substitute for formylation of methionyl tRNA for folateless protein initiation.