The penetration of [3H]thymidine, [3H]D-leucine, [125I]albumin, and the drugs [3H]5-fluorouracil and [3H]vinblastine into human glioma spheroids (in vitro tumor models) was studied by a method based on rapid freezing, freeze drying, vapor fixation, wax embedding, dry sectioning, and contact autoradiography. No significant disturbances in the distribution of water soluble substances were observed. Thymidine and D-leucine penetrated the whole spheroids relatively fast, whereas albumin showed reduced penetration. The concentration of albumin was highest at the periphery of the spheroids, but only smaller amounts were detected in the deeper regions. A significant difference between the penetration patterns of the drugs studied was also observed. Fluorouracil penetrated rather freely, but the penetration of vinblastine was limited.