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Two presumptive single-step mutants, resistant to penicillin and sensitive to the bactericidal effect of normal human serum, were isolated on penicillin gradient plates from a smooth, penicillin-sensitive, serum-resistant strain of Salmonella enteritidis(O9,12; gal hisEI cys). The chemical composition of their lipopolysaccharides, their phage-sensitivity patterns, and their serological and cultural properties showed one to be ‘part-rough’ and the other smooth. Hfr strains with O antigens O4,5,12 or 01,4,5,12 (two Salmonella typhimurium and one S. abony) and one S. enteritidisF′, O9,12, were crossed with the S. enteritidisO9,12 mutants. The results with the part-rough mutant indicate that its penicillin-resistance, serum-sensitivity and rough phage pattern result from a single mutation between hisEI and the part of the rfbgene cluster determining O specificity, 4 (abequose) or 9 (tyvelose). Transduction experiments confirmed that the mutation is closely linked to the hisoperon. This mutation is inferred to cause an incomplete defect in a transferase for galactose, mannose or rhamnose, the smooth sugars common to O4,5,12 and O9,12. Results from similar crosses to the smooth, serum-sensitive, penicillin-resistant S. enteritidismutant indicate that its serum-sensitivity is not linked to his.The occasional independent segregation of penicillin-resistance and serum-sensitivity suggests that other loci modify penicillin- resistance.