Trimethoprim (TMP), an inhibitor of dihydrofolate reductase, decreases the level of tetrahydrofolate supplying one-carbon units for biosynthesis of nucleotides, proteins, and panthotenate. We have demonstrated for the first time that one of the effects of the TMP action in E. coli cells is protein aggregation and induction of heat shock proteins (Hsps). TMP caused induction of DnaK, DnaJ, GroEL, ClpB, and IbpA/B Hsps. Among these Hsps, IbpA/B were most efficiently induced by TMP and coaggregated with the insoluble proteins. Upon folate stress, deletion of the delta ibpA/B operon resulted in increased protein aggregation but did not influence cell viability.