The in vitro activities of three new 8-methoxychlortetracyclines, Sch 36969, 33256 and 34164 were compared to tetracycline, minocycline and doxycycline. Against aerobic Gramnegative rods Sch 36969 had a geometric mean MIC (GMM) of 4.2 μg/ml, about 8-fold more potent than Sch 33256, and similar to all the other compounds. Sch 36969 also had good activity against methicillin-resistant (GMM, 0.21 μg/ml) and -susceptible Staphylococci (GMM, 0.14 μg/ml), Streptococci (GMM, 0.06 μg/ml), and most anaerobic bacteria (GMM, <0.5 μg/ml). In general, Sch 36969 was similar to, or more potent than, all the other compounds tested. Serum levels of Sch 36969 in squirrel monkeys were 4-fold lower (AUC, 4.5 μg•hours/ml) than those of chlortetracycline (AUC, 16.1 μg•hours/ml). In mouse protection tests (PD₅₀s) against various strains of bacteria, Sch 36969 was similar in activity to tetracycline, but up to 6-fold less active than chlortetracycline. The structure activity relationships for these new chlortetracyclines are described.