Crotonaldehyde is an alpha,beta-unsaturated carbonyl compound and an important environmental and industrial toxic substance. Its mutagenic and carcinogenic properties are related to its reactivity to DNA, where it forms different guanine adducts. In order to study the mutagenic consequences of this agent in intact human cells, we treated the shuttle vector plasmid pZ189 with different doses of crotonaldehyde at 37 degrees C for 2 h and then transfected the such damaged plasmid into the normal human lymphoblast cell line GM0621. Within these host cells the guanine adducts are repaired and the plasmids replicated by cellular enzymes. After 2.5 days replicated plasmids were purified from the cells and plasmid survival was quantitated by transformation ability. With increasing doses of crotonaldehyde, we found a significant decline of plasmid survival, reflecting a pronounced genotoxicity of crotonaldehyde-induced DNA damage in intact human cells. Using the plasmid encoded mutagenesis marker gene supF, we were able to screen for mutants and determine mutation frequency in recovered plasmids. A significant increase in mutation frequency with increasing doses of crotonaldehyde reflects mutagenicity of crotonaldehyde-induced DNA damage. Base sequence analysis of recovered mutants revealed 39% point mutations, 46% deletions, and 15% insertions and inversions. Most of the point mutations (82%) were located at G:C base pairs, which is well explained by the DNA damage profile of crotonaldehyde. Among deletions we found a frequent reoccurrence of two hot spot deletions, representing 62% of all deletions. The sites of breakpoints of these deletions hot spots and of other deletions within the plasmid were also found to be sites of DNA breaks, directly induced by crotonaldehyde, as seen in an endlabeled plasmid fragment, treated with crotonaldehyde. Further analysis of the flanking sequences around the deletion breakpoints revealed a high frequency of four different kinds of short sequence homologies of up to eight base pairs.