Cloxacillin and flucloxacillin were administered to horses by single intravenous dose (25, 33 or 44 mg/kg). Levels of these compounds were assayed biologically from serum and synovia of the carpal and tibio‐tarsal joints at intervals up to 360 min. Serum levels of cloxacillin at 30 min were all in excess of 48 μg/ml and were equal to, or higher than, those given by the equivalent doses of flucloxacillin. Highly significant differences (P<0.001) between doses and between sampling times (30, 60, 90, 120, 180, 240, 360 min) were detected for both compounds. Although the rates of elimination of cloxacillin and flucloxacillin from blood did not differ significantly, the early establishment of higher levels of cloxacillin in blood meant that it regularly persisted longer than flucloxacillin in both serum and synovia. The rate of elimination from serum was significantly steeper (P<0.001) than that computed for synovia. The slower rate of loss of cloxacillin or flucloxacillin from the synovia than the blood appears to fit the model of transfer between serum and synovium proposed by Howell, Sutherland&Rolinson (1972). The persistence of cloxacillin and flucloxacillin in synovia beyond the time when effective minimum inhibitory concentrations (4 μg/ml) were present in the blood, indicates a practical advantage in equine therapeutics where septic arthritis is the major indication for use of these compounds. In these situations, cloxacillin has a further clinical advantage in persisting in synovia for a longer time than flucloxacil