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This study tested the hypothesis that increasing the duration and/or frequency of antimicrobial treatment of subclinical mastitis would result in a higher bacteriological cure rate. Glands with a positive California mastitis test (CMT) from cows with an elevated somatic cell count (>500,000 cells/mL) that had an intramammary infection were randomly assigned at cow level to no treatment (Control; <i>n</i> = 80 glands), intramammary infusion of 200 mg cloxacillin sodium on three occasions at 48 h intervals (3 × 48 h; <i>n</i> = 273 glands), five occasions at 24 h intervals (5 × 24 h; <i>n</i> = 279 glands), or on five occasions at 48 h intervals (5 × 48 h; <i>n</i> = 72 glands). Glands were resampled at 21 (±3) and 28 (±3) days after initiation of treatment. The gland-level cure rate for any pathogen was 5/80 (6.2%), 139/173 (49.8%), 172/297 (61.6%) and 58/72 (80.6%) for Control, 3 × 48 h, 5 × 24 h and 5 × 48 h, respectively. The cure rate for major pathogens (defined as <i>Staphylococcus aureus</i> or <i>Streptococcus</i> spp.) was 4/52 (7.7%), 84/197 (42.6%), 96/183 (52.5%) and 36/48 (75%) for Control, 3 × 48 h, 5 × 24 h and 5 × 48 h, respectively. We conclude that treatment was superior to no treatment, and bacteriological cure rate was higher with the 5 × 24 h protocol than for the 3 × 48 h protocol and was higher with the 5 × 48 h than the 5 × 24 h protocol.