Under proper conditions, one infective center was obtained for 3 x 10(8) molecules of P22 phage deoxyribonucleic acid (DNA) when lysozyme-ethylenediaminetetraacetic acid spheroplasts of Escherichia coli were transfected in the presence of 25 mug of protamine sulfate per ml. A 3- to 50-fold B-specific and K-specific E. coli restriction of the incoming P22 DNA was observed. When P22 DNA-infected E. coli spheroplasts were plated with infertile r(LT) (+)m(LT) (+)Salmonella typhimurium indicator, an additional 70-fold restriction was observed. In the presence of protamine sulfate, penicillin spheroplasts of S. typhimurium SB1330 could be transfected b P22 DNA with efficiencies sometimes approaching those obtained with the E. coli spheroplasts; thus, facilitation of transfection by protamine sulfate is not limited to E. coli or to lysozyme-ethylenediaminetetraacetic acid spheroplasts. The application of these results to studies of transfection among other genuses and to studies of in vitro host-controlled restriction and modification for the two loci in S. typhimurium and the one locus in E. coli is discussed.