Earlier cytokinetic studies using 9L monolayer culture cells showed that cells began accumulating in the G2 phase (4C position in the DNA distribution) within 30 hr after administration of 1,3-bis(2-chloroethyl)-1-nitrosourea (3 micrograms/ml). Most of these cells reached the G2 phase at 48 hr and remained there for another 24 hr. Similar studies using 9L spheroid culture cells show that 9L cells also began to accumulate at the 4C DNA position 30 hr after administration of 1,3-bis(2-chloroethyl)-1-nitrosourea (3 micrograms/ml); however, only one-half of the cells accumulated in the 4C peak during the first 48 hr after treatment, while the other half remained at the 2C position. Those that remained at the 2C peak might correspond to the G0 cells that exist in 9L spheroids. When one-half the cells were in 2C and one-half were in 4C, the spheroids were dissociated using an enzyme cocktail and sorted using centrifugal elutriation. Cells from each elutriated fraction were analyzed for DNA content using flow cytometry and for viability using a colony-forming efficiency assay. Results indicated that in spheroids, 1,3-bis(2-chloroethyl)-1-nitrosourea (3 micrograms/ml) is more effective against noncycling cells and that repopulation is more vigorous in G2-arrested cells, which presumably were cycling at the time of treatment.