The effect of mitomycin C on the synthesis and degradation of DNA in mouse P-388 cells grown in suspension culture has been investigated. Treatment for 24 hours with 0.5 μ/ml of the antibiotic selectively suppressed DNA synthesis in these cells. DNA synthesis was measured as incorporation of 3H-TdR, and during the first 2.5 hours of treatment the only effect of mitomycin C on this parameter was a reduced incorporation on the DNA polymerization level. However, more than 8 hours after exposure to the antibiotic, reduced uptake of this precursor into the cold acid soluble pool was found in the treated cells. Exposure for 1 hour to 0.5 μg/ml of mitomycin C also caused a slightly reduced rate of DNA synthesis initially, but in these cells no significant decrease in total DNA was found 24 hours after the treatment. Mitomycin C-induced degradation of DNA was not found during the first 24 hours of treatment.