Melphalan resistance developed previously in a human melanoma cell line (MM253) could not be further increased. Cross-resistance was found to nitrogen mustard but not to ultraviolet light radiation. A clone of MM253 had the same drug sensitivity and heterogeneous chromosome complements as did the parent culture. The melphalan-resistant cells (MM253-12M) had 2.6-fold the D0, 1.5-fold the size, 1.3-fold the RNA content, 1.4-fold the protein content, and 2.6-fold the DNA content of the sensitive parent line. There was no evidence for activation or detoxification of melphalan by intact melanoma cells or by mouse liver microsomes competent for the activation of other drugs. Melphalan transport was similar in both cell lines, reaching a steady-state level 3 times the concentration in the medium after 2.5 min. Both lines covalently bound the same total amount of [3H]melphalan per cell, but in MM253-12M a 50% decrease in binding to DNA was almost sufficient to account for the increase in resistance. The level of melphalan-induced DNA interstrand cross-links, which were heat labile but not alkali labile, reached a maximum during the 4-hr treatment period and then declined slowly. The degree of cross-linking in MM253-12M was 50% less than that in MM253. Unlike ultraviolet light, methyl methanesulfonate, and nitrogen mustard, melphalan at equitoxic doses did not damage the DNA sufficiently to immediately inhibit DNA synthesis. Although both lines were proficient for repair of ultraviolet light and methyl methane sulfonate damage, melphalan did not induce significant levels of DNA repair synthesis and had little effect on the rate of DNA chain elongation. In MM253 cells, strand breaks were detected only at high melphalan doses; MM253-12M formed breaks more readily. This evidence suggests that the cross-linking events and that developed resistance arises from decreased susceptibility to DNA to this damage.