As part of an effort to develop an animal model for studies of uptake of human beta-glucuronidase, fibroblasts were established from primary explants of connective tissue from nine different animal species, and examined for their ability to take up human platelet beta-glucuronidase. Endogenous fibroblast beta-glucuronidase was inactivated by heating extracts to 65 degrees for 30 min. Human beta-glucuronidase was stable to this treatment. Uptake of human beta-glucuronidase by animal fibroblasts was measured as heat-stable beta-glucuronidase present in fibroblasts after exposure to partially purified human platelet beta-glucuronidase for 48 hr. Althought all animal fibroblasts examined exhibited some uptake capacity for human beta-glucuronidase, the uptake capacity of different animal fibroblasts varied over a 10-fold range. The uptake capacity of bovine fibroblasts was at least 80% that of human fibroblasts. Rat and hamster fibroblasts showed about half the uptake capacity of human fibroblasts. The rat fibroblasts resembled the human fibroblasts in the kinetics of uptake of hihg uptake (platelet) enzyme, poor uptake of human placental enzyme, and lack of appreciable turnover of enzyme taken up over 4 days. Heating extracts of rat organs containing added human beta-glucuronidase at 65 degrees selectively inactivated rat enzyme.