Infections due to Shiga toxin-producing Escherichia coli (STEC) are responsible for severe diarrheal diseases in humans, and these bacteria have recently emerged as a leading cause of renal failure and encephalitis in children and the aged. In this study, we examined the environment-dependent production of proteins secreted from a strain of STEC O26:H11 by trichloroacetic acid precipitation, SDS-PAGE, Western blotting and N-terminal amino acid sequence analysis. Growth of bacteria in essential minimum medium (M9) led to the detection of secreted proteins of 104, 80,40, 37 and 25 kDa (P104, P80, P40, P37 and P25, respectively). When grown in serum-free MEM, only P104, P40, P37 and P25 were observed in supernatant fluids. Growth of the bacteria in Luria-Bertani broth (LB) enhanced the expression of P104, but the productions of the other proteins were remarkably reduced. CO2 increased the secretion of P80 and P37, but reduced the production of P104. N-terminal amino acid sequencing revealed that P104 was EspP of STEC, which was homologous to EspC of enteropathogenic Escherichia coli (EPEC), and both proteins belong to a subclass of the IgA protease family. P80, which was identified as EspE of STEC, was homologous to Tir of EPEC. P40, P37 and P25 were found to be highly homologous to the similarly sized EspD, EspB and EspA proteins, previously detected in culture supernatants of EPEC. Those proteins are thought to be STEC virulence factors. Sera were obtained from two patients, one with colitis and another with hemolytic uremic syndrome (HUS), caused by STEC O157:H7, to study immune response to secreted proteins. Our results suggested that Tir caused immune response following STEC disease.