Theophylline tissue distribution was examined in normal and dietary-induced obese Sprague-Dawley rats following constant infusion to steady-state. Theophylline distribution was linear among all tissues and uniform throughout body water spaces. The apparent distribution coefficient, Kd,app (tissue: serum concentration) ranged from 0.55 to 0.69 for all lean tissues, but averaged 0.08 in fat. Obese rats had a consistently higher Kd,app in several tissues including muscle, heart, and liver caused by reduced (from 42 to 33 per cent) serum protein binding. Muscle slice uptake of theophylline from buffer was similar in normal and obese animals, confirming serum binding as the factor altering Kd,app. The conceptual and applied bases of calculating and measuring steady-state volume of distribution (VSS) by various methods were explored. The physiological perturbations of theophylline VSS by obesity can be accounted for by alterations in serum binding and expanded lean and fat tissue masses, rather than changes in tissue partitioning.