The effect of radiation fractionation was investigated using a new 96-well-plate clonogenic assay in four squamous cell carcinoma lines. Earlier experiments had shown that two of the cell lines (UT-SCC-1A and UM-SCC-14A) were inherently relatively sensitive to irradiation, and two (UM-SCC-1 and UM-SCV-1A) relatively resistant. All of the four carcinomas from which the cell lines were established had poor clinical outcome. The radiation doses were given as a single exposure, or split into two or three equal fractions with a 24-h interval. The two inherently sensitive cell lines showed enhanced survival after radiation fractionation as compared with a single dose, whereas the resistant cell lines did not. The result suggests that both the inherent resistance of cancer cells to irradiation and the repair of sublethal radiation damage may lead to treatment failure, and that shortening of the total irradiation time may overcome cancer cell recovery between fractions in some, but not in all carcinomas.