We have examined the in vitro sensitivity to Adriamycin of human ovarian cancer colonies cloned in soft agar. In the 26 patients tested, 3 different patterns of sensitivity to Adriamycin were observed: (a) in 75% of the previously untreated patients, there was greater than 70% reduction in colony-forming cells after exposure to Adriamycin (1.0 micrograms/ml), a level which approximates the peak plasma level after i.v. therapy; (b) in all the patients who had progressive disease while on a chemotherapy regimen without Adriamycin, a greater than 70% reduction in colony-forming cells was observed only at a concentration of 10 micrograms/ml, a level not achievable by i.v. administration; (c) in 80% of patients with progressive disease after treatment with Adriamycin as part of the primary chemotherapy regimen, a 70% reduction in tumor colony-forming cells could not be achieved even at 10 micrograms/ml. These in vitro results are in agreement with clinical observations regarding the effectiveness of Adriamycin in previously untreated patients (42% response rate) with ovarian cancer as well as its ineffectiveness (0 to 6% response rate) as a second-line therapy in relapsed patients. The results also have provided a rationale for an ongoing Phase I trial of i.p. Adriamycin in patients with ovarian cancer from Group b above since cytotoxic levels can be produced i.p. using large-volume dialysis via a Tenckhoff dialysis catheter. The relative cytotoxicity of Adriamycin to its two major metabolites, adriamycinol and adriamycin aglycone, was also determined in the clonogenic assay. Both derivatives produced suppression of ovarian cancer colony formation; however, Adriamycin was more cytotoxic that was either metabolite.