In Escherichia coli B grown at 37 °C, essentially all spontaneous deletions detected by simultaneous mutation to resistance to the bacteriophage T1 and to tryptophan auxotrophy ( tonB trp del mutations) remove the entire trp operon (B-type deletions). In contrast, tonB trp del mutations in E. coli K12 have a more random assortment of end points terminating within any of the five structural genes (K-type deletions) or beyond them. Chromosomal hybrids, constructed by transduction of the trp region from E. coli B into E. coli K12 (KB hybrids), could be divided into two distinct classes on the basis of their deletion patterns; some produced K-type deletions with the same relative frequency as in strain K12, while the others all produced the B-type and, without exception, at a 10- to 20-fold higher relative frequency than that observed in strain B. Further analysis of KB hybrids showed that the chromosomal region from E. coli B approximately midway between the cysB and trp operons is the region responsible for the generation of the B-type deletions characteristically found in this strain. Evidence was obtained suggesting that the end points of the B-type deletions are not at fixed points in the chromosome. TonB trp deletions occurred in strains K12(W3110), B and in the KB hybrids at 30, 37 and 42 °C, but in all these strains the relative frequency of deletions was reduced at 42 °C more than sevenfold; the most pronounced reduction, about 40-fold, was observed for the B-type deletions occurring in the KB hybrids. Protein synthesis at 37 °C is required for production of the B-type deletions at the high frequency. The reduction in the generation of deletions when the cells are grown at 42 °C may be due to temperature sensitivity of a protein. DNA replication is probably not required for the event that culminates in a B-type deletion since, under certain conditions, deletion events occur in a thymidine auxotroph apparently in the absence of thymidine. The effects of thymidine starvation, however, are complicated and depend on whether a preceding period of amino-acid starvation is allowed. The bacterial recA and recB functions are not required for the production of the B-type deletions.