3'-(3-Cyano-4-morpholinyl)-3'-deaminoadriamycin (CMA) is a highly potent analogue of the antitumor agent, Adriamycin (ADR), being up to 1500 times more cytotoxic both in vivo and in vitro. In contrast to ADR, CMA, and 5-imino-3'-(3-cyano-4-morpholinyl)-3'-deaminoadriamycin (ICMA) have been shown to possess alkylating activity, as seen by their ability to produce DNA-DNA cross-links in human and murine tumor cells and in isolated lambda-phage DNA. We have compared the pharmacological activities of CMA, ICMA, and the alkylating agent, chlorambucil (CHL), in order to determine the roles of intercalation, the quinone ring, and DNA base composition, in cross-linking by CMA. CMA was 27-and 1000-fold more active than ICMA and CHL, respectively, in cross-linking DNA in L5178Y cells. In addition, the maximum level of cross-linking in L5178Y cells was reached more rapidly with CMA than with CHL, and the CMA cross-links were removed faster and more efficiently by these cells. CMA was 26- and 450-fold more active than ICMA and CHL, respectively, in producing DNA cross-links in isolated lambda-phage DNA. In contrast, the alkylating activity of CMA was only 6-fold greater than CHL, as measured by the ability of the drugs to bind to the nucleophile, p-nitrobenzyl pyridine. CMA was a better DNA intercalator than ICMA, whereas CHL did not intercalate. In addition, the intercalating agent, ethidium bromide, inhibited the cross-linking activity of CMA, but not that of CHL, suggesting that intercalation contributed to the cross-linking activity of CMA. CMA produced an increasing level of cross-linking, but showed no difference in intercalation, with isolated DNA of increasing G-C content, suggesting a preference for alkylating G-C bases. Both the cross-linking and intercalating, but not the alkylating, activities of CMA and ICMA were decreased by the reducing agent, sodium borohydride, providing additional evidence that the intercalative interaction of the ADR analogues with DNA contributes to their DNA cross-linking activity. Thus, alterations to the quinone group may effect the intercalating activity of these analogues and may contribute to the difference in cross-linking activity between CMA and ICMA.