Chemotherapeutic agents are used in increasingly high dosages to treat patients with refractory cancers. An in vitro clonogenic assay was used to investigate the effects of melphalan on cultures of human neuroblastoma, Ewing's sarcoma, and osteosarcoma cells, as well as on normal bone marrow cells. A 1-hr incubation with 10(-5) M melphalan significantly inhibited tumor colony growth of both fresh neuroblastoma and osteosarcoma cells (p less than 0.01) while Ewing's sarcoma cells and normal bone marrow appeared resistant to melphalan even at a 100-fold higher concentration. Incubation with melphalan for 8 hr did not significantly increase the sensitivity of neuroblastoma and osteosarcoma or the relative resistance of Ewing's sarcoma cells; however, normal bone marrow which had remained resistant to melphalan after 1 hr of incubation, showed significant inhibition of colony growth after an 8-hr incubation with the agent. Repeated exposure to melphalan increased the degree of inhibition of both tumor and normal marrow colonies. Fresh neuroblastoma cells were significantly more sensitive than long-term cultured neuroblastoma cells at all drug doses tested. HPLC studies demonstrated that the loss of melphalan followed first-order kinetics with a half-life of 69 min, and that in addition to the 2 known breakdown products, mono- and dihydroxy-melphalan, several other peaks were present which were not attributable to the tissue culture medium or the buffer solutions.