In order to achieve a better understanding of factors involved in drug penetration into poorly vascularized tumour tissue, the penetration of some model substances was studied in vitro. Multicellular human tumour spheroids were used as model system. The test substances were [3H]thymidine and [14C]glucose, both of which are capable of passing easily through cell membranes, and [3H]thymidine-5'-triphosphate, [3H]sucrose and [3H]inulin, all of which are unable to pass directly through cell membranes. The penetration of these substances was studied using a dry histological and autoradiographical method preserving the distribution of water-soluble substances. The two thymidine compounds penetrated very efficiently into the spheroids, and their penetration patterns were rather similar. The saccharides differed somewhat in their penetration properties. Glucose had the fastest penetration and inulin the slowest. After 15 min, however, inulin was also found isotropically distributed within the spheroids. Thus, extracellular penetration seemed to be a possible way for a substance to reach the central parts of a spheroid. The differences between the saccharides could be due to some extent to differences in molecular weight and solubility.