We investigated the induction of a stress response gene by anticancer drugs that damage and covalently modify DNA and other cellular macromolecules. Two human colon adenocarcinoma cell lines (HT-29 and BE) which differ in sensitivity to chloroethylnitrosoureas were treated with 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) or with 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU). Both of these drugs can alkylate, crosslink and carbamoylate cellular macromolecules. Treated cells were compared to controls for cytoplasmic levels of HSP70 RNA and for synthesis of heat shock proteins. We also tested for induction of stress response gene expression by equitoxic or greater concentrations of other nitrosourea analogues which can alkylate only, alkylate and crosslink only or carbamoylate only, as well as other DNA crosslinking agents (chlorambucil and cis-platinum). Of these, only BCNU and CCNU, the chloroethylnitrosoureas having all three of the macromolecule-modifying activities, strongly induce HSP70 RNA levels in a dose-dependent and time-dependent manner. Induction of HSP70 by BCNU occurred in both cell lines at dose ranges that were cytocidal to the BCNU-resistant HT-29 cells. No induction was seen in BE cells at the lower BCNU concentrations that were equitoxic to that more sensitive cell line. These observations suggest that induction of HSP70 by BCNU and CCNU is neither a direct consequence of DNA crosslinks nor an invariable result of cytocidal drugs.