Using [14C] benzyl penicillin, we determined the ability of penicillin to cross capillary membranes in the tibiae of dogs, the degree of protein-binding of penicillin in serum and tibial bone tissue, and the concentration of penicillin in the interstitial fluid space of tibial bone. Extraction studies demonstrated that the capillary membranes in bone were readily traversed by benzyl penicillin. Penicillin concentrations in filed cortical and crushed cancellous bone samples, measured by both isotopic and biological assays, ranged from 1.0 to 8.7 per cent of the simultaneous concentrations of penicillin in plasma. Twenty-one per cent of the penicillin in plasma was protein-bound, while 39 per cent of the penicillin in cortical bone was tissue-bound and not biologically active. Studies of the distribution of benzyl penicillin showed that it was distributed in equal concentrations in the plasma and in the interstitial fluid spaces of bone. The concentration of biologically active penicillin in the interstitial fluid spaces of bone correlated closely with the simultaneously observed plasma level in animals in which a steady-state equilibrium had been achieved.