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We analyze experience with 600 specimens for in vitro chemosensitivity assessment of human neoplasms utilizing a soft agar colony-forming technique. Good test reproducibility is demonstrated. Disaggregation with collagenase enhances yield and does not alter chemosensitivity profiles. Therapeutic exposure to chemotherapy prior to biopsy reduces in vitro sensitivity to the specific agents used in vitro. The cyclophosphamide derivatives 4-hydroperoxycyclophosphamide (4-HC) and phosphoramide mustard are active in vitro, and produce comparable rank order sensitivities among tested tumors. There is marked reduction of in vitro 4-HC sensitivity in patients with prior therapeutic cyclophosphamide exposure, supporting the use of this derivative in test systems. Rank order of test results among specimens is compared at 0.1 µg and 10 µg drug/ml. Substantial differences in rank order at these two dose levels are demonstrated, indicating that the in vitro test dose selected is an important variable.