The Escherichia coli K-12 gene coding for the nucleoid-associated protein HNS was cloned together with 5.6 kb of downstream DNA in the vector pACYC184. The cloned DNA complemented a mutation in the osmZ locus of E. coli, which codes for HNS. However, the multicopy plasmid harboring the cloned sequence was found to be mutagenic and to produce at high frequency mutations that mapped to the E. coli cya gene, which codes for adenylate cyclase. Acquisition of the cya mutations was independent of RecA. These mutations were phenotypically suppressed by providing the cells with exogenous cyclic AMP and were complemented in trans by a plasmid carrying an active copy of the cya gene. A deletion analysis of the cloned sequences showed that DNA downstream of the gene coding for HNS was also required for the mutagenic effect of cya and had a role in regulating the expression of the osmZ-dependent proU locus. These sequences appear to contain at least two genetically active regions.