Background and purpose: The objective of the present study was to evaluate the predictive potential of cell kinetic parameters and repopulation rates determined by flow cytometry during multifraction irradiation of spheroids, a system in which the fate of all cells can be determined with high precision. Ultimately, similar analytical techniques should provide a reproducible and prognostically significant clinical predictive assay.Materials and methods: Multicellular spheroids of Chinese hamster V79 lung cells were irradiated with 2.5 Gy of 250 kVp X-rays twice daily to a total dose of 25 Gy. Repopulation parameters and cell kinetic parameters were followed throughout the irradiation period and for 5 days after completion of exposure.Results: (1) Regrowth (RG) took place early during multifraction irradiation. (2) Potential doubling time (T p o t ) decreased steadily from the early part of treatment, remaining of short duration until the spheroids almost attained the pre-treatment number of clonogenic cells. (3) Accelerated repopulation was mainly due to a decreased cell loss factor (Φ) and increased growth fraction (GF), although a modest decrease in cell cycle time (t c ) was suggested. (4) Φ decreased during exponential RG. (5) Other parameters such as observed doubling time (t d ) and labelling index (LI) paralleled these findings.Conclusions: Clonogen repopulation that began early in the irradiation scheme and accelerated rapidly is not consistent with the prevailing view that accelerated repopulation begins several weeks into clinical protocols. Also, pre-treatment T p o t did not adequately estimate the repopulation speed in the spheroids. Equivalent studies in animal tumour systems, and then in the clinic, are consequently indicated and of some urgency.