A microcolony assay was used in conjunction with fractionated gamma irradiation to determine the number of clonogens in murine intestinal crypts with varying doses of irradiation used in the determination. The experimental design allows direct comparison between two-dose methodologies, employing one and two (or two or four) equal dose fractions, and multiple-dose methodologies involving determination of the crypt survival curves for a number of fractionation regimens using equal doses per fraction. The two-dose methodology yielded estimates of clonogen number of between 3 and 4 at low delivered dose (single and double fractions each of 6.5-7.5 Gy), rising to around 40 at high biological doses (two and four fractions each of 5.75 or 6.5 Gy). The multifraction methodology yielded estimates of clonogen number which increased from 13 after a single fraction to values of 26 and 22 after three and four fractions. However, the latter values were reduced to 11 and 9, and showed little evidence of any dependence on fraction number, when data pertaining to high biologically effective doses were excluded. Hence it is concluded that the high values for clonogen number typically deduced from such multiple-dose protocols, compared with the generally lower (but dose-dependent) values obtained from two-dose protocols, may be explained at least partially by the higher biological doses generally employed in the multiple-dose protocols.