Chlamydia pneumoniae is an important obligate intracellular pathogen that replicates within an inclusion in the eukaryotic cell. The initial event of a chlamydial infection is the adherence to and subsequent uptake of the infectious elementary bodies (EBs) by the human cell. These processes require yet-unidentified bacterial and eukaryotic surface proteins. The GroEL1 protein, which exhibits a very strong antigenicity and in vitro can activate various eukaryotic cells, is a potential pathogenicity factor. We localized the protein during the infection process and found it in the inclusion but outside the chlamydial particles. GroEL1 was also localized on the surface of EBs, and the protein could be washed off the EBs. Latex beads coated with recombinantly produced GroEL1 (rGroEL1) bound in a dose-dependent manner to HEp-2 cells. Likewise, GroEL1, when expressed and displayed on the yeast cell surface, mediated adhesion to HEp-2 cells. Interestingly, the homologous GroEL2 and GroEL3 proteins showed no adhesive properties. Incubation of primary umbilical vein endothelial cells with soluble GroEL1 and GroEL1-coated latex beads activated the translocation of the general transcription factor NF-kappaB into the nucleus. Finally, preincubation of HEp-2 cells with rGroEL1 significantly reduced subsequent infection with C. pneumoniae, although adhesion of infectious bacteria to eukaryotic cells was not affected. Taken together, these data support a role for extracellular GroEL1 in the establishment of the chlamydial infection.