Most organisms respond to heat by substantial alteration of the pattern of gene expression. This has been particularly well studied with Escherichia coli although the response has by no means been completely characterized. Here we report the characterization of 26 new heat shock genes of E. coli, termed hsl, discovered by global transcription analysis with an overlapping lambda clone bank. We have measured the molecular weights of the corresponding heat shock proteins and mapped each of them to within a few kilobases on the E. coli genome. In vitro, 16 of them can be activated by the E sigma 32 RNA polymerase, which specifically transcribes heat shock genes. In vivo expression kinetics of seven of eight examined new proteins were found to be similar to those of the four most studied heat shock proteins, DnaK, DnaJ, GroEL (MopA), and GroES (MopB). In the course of this work, we confirmed that the catalytic subunit of the ATP-dependent Clp protease (also known as Ti protease), ClpP, is derived from a larger precursor protein. Possible assignments of some of the hsl genes to known proteins are discussed.