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Enterococci exhibit two types of resistance to streptomycin. Moderately highlevel resistance is observed in most naturally occurring strains and can be overcome by simultaneous exposure to penicillin. In addition, very high-level resistance is found in those strains against which penicillin plus streptomycin fail to produce synergism in vitro. To study the mechanism of streptomycin resistance in enterococci, ribosomes from a wild-type strain and from a highly streptomycin-resistant mutant were isolated, characterized, and studied in an in vitro amino acid incorporation system. The ribosomes from the organism with moderately high-level streptomycin resistance were sensitive to streptomycin in vitro, suggesting that this type of resistance is caused by failure of streptomycin to reach the ribosomes. Very highlevel resistance (and lack of penicillin-streptomycin synergism), on the other hand, appears to be due to ribosomally mediated streptomycin resistance. Most strains of enterococci are resistant to streptomycin by conventional antibiotic sensitivity testing (22). The addition of penicillin overcomes this resistance (i.e., produces synergism with streptomycin) in many but not all strains of