Most organs have capillary beds which are porous, permitting antibiotics to pass into the extravascular fluid with ease. Two major factors determine the levels of free (unbound) antibiotic in tissue fluids: the levels of free drug in the serum and the length of the diffusional paths. When the latter are short, for example in ‘interstitial fluid’, the levels of free drug in the extravascular fluid closely parallel the levels of free drug in the serum. In contrast, when diffusional paths are long, for example in large reservoirs or tissue cages, the levels of free drug in the center of the collection approximate the logarithmic mean of the peak and trough levels of free drug in the serum. The concentrations of total drug in these sites exceed the concentrations of free drug by the amount that is bound to serum albumin or tissue components. If drug is inactivated at local sites of infection more rapidly than it can enter, the levels will be lower than predicted from the equilibrium relationships described above. In ‘specialized sites’ such as the central nervous system, vitreous humor, and prostate gland, the capillary beds are non-porous; as a result, lipid-solubility of the antibiotic is a crucial determinant of penetration. Serum protein-binding plays a lesser role in this situation. Active transport reduces the levels of beta-lactam antibiotics in the central nervous system and vitreous humor, but produces high levels of these agents in excretory sites such as the proximal renal tubular cell and bile. Knowledge of these relationships permits the physician to estimate the levels of bioactive drug in tissue fluids on the basis of serum concentrations.