The ability of intracellular pathogens to sense and adapt to the hostile environment of the host is an important factor governing virulence. We have sequenced the operon encoding the major heat shock proteins GroES and GroEL in the gram-positive food-borne pathogen Listeria monocytogenes. The operon has a conserved orientation in the order groES groEL. Upstream of groES and in the opposite orientation is a gene encoding a homologue of the Bacillus subtilis protein YdiL, while downstream of groEL is a gene encoding a putative bile hydrolase. We used both reverse transcriptase-PCR (RT-PCR) and transcriptional fusions to the UV-optimized Aequorea victoria green fluorescent protein (GFP(UV)) to analyze expression of groESL under various environmental stress conditions, including heat shock, ethanol stress, and acid shack, and during infection of J774 mouse macrophage cells. Strains harboring GFP(UV) transcriptional fusions to the promoter region of groESL demonstrated a significant increase in fluorescence following heat shock that was detected by both fluorimetry and fluorescence microscopy, Using both RT-PCR and GFP technology we detected expression of groESL following internalization by J774 cells. Increased intracellular expression of dnaK was also determined using RT-PCR, We have recently described a system which utilizes L. monocytogenes hemolysin as an in vivo reporter of gene expression within the host cell phagosome (C, G, M, Gahan and C, HiII, Mel. Microbiol. 36:498-507, 2000), In this study a strain was constructed in which hemolysin expression was placed under the control of the groESL promoter, In this strain hemolysin expression during infection also confirms transcription from the groESL promoter during J774 and murine infection, albeit at lower levels than the known virulence factor plcA.