Hamster fibrosarcoma cells were synchronized by mitotic selection and exposed to varying concentrations of 1-β-d-arabinofuranosylcytosine (ara-C) for 2 hr in mid-S phase. There was a direct relationship between DNA synthesis inhibition and cytotoxicity produced by ara-C once DNA synthesis was decreased by over 85%. The noncytotoxic concentration of 10 -5 m ara-C produced little chromatid breakage; but extensive chromatid breakage and chromosomal rearrangement were seen in cells treated with the cytotoxic concentration of 10 -3 m ara-C, thus supporting earlier observations that chromatid breakage is highly correlated with cytotoxicity. Predominantly small DNA was synthesized when cells were treated with both 10 -5 and 10 - 3 m ara-C, and this DNA could be completely chased into high-molecular-weight DNA after addition of deoxycytidine. Both concentrations of ara-C also inhibited, to different degrees, the joining of intermediate-size DNA fragments into larger DNA; thus neither parameter appeared directly related to the ara-C-produced cytotoxicity. © 1976, American Association for Cancer Research. All rights reserved.