The effects of the two antitumor drugs vinblastine and 5-fluorouracil on the growth of the human tumor cell lines U-118 MG (glioma) and HTh-7 (thyroid cancer) were analyzed. The cells were cultured both as monolayers and as multicellular spheroids and exposed to vinblastine (0.1, 1.0, or 10 micrograms/ml) or 5-fluorouracil (10, 100, or 1000 micrograms/ml) for 15 min, 2 hr, or 24 hr. The drugs induced growth delays of the monolayers and delays in the outgrowth of cells from spheroids which were placed on cell-adhesive surfaces. Cell cultures exposed to sublethal drug doses showed a dose-dependent lag period followed by regrowth at normal growth rates. In all cases with vinblastine exposures, the spheroids seemed more resistant to the drug treatments than did the monolayer cultures. Much smaller differences were obtained after treatments with 5-fluorouracil. The three-dimensional arrangement of cells in spheroids giving rise to, e.g., nutrient and proliferation gradients may, to some extent, be responsible for the increased resistance. The spheroids were especially resistant to short treatments with vinblastine. This was probably due to penetration barriers.