Cytolysin-mediated translocation (CMT) is a recently described process in the Gram-positive pathogen Streptococcus pyogenes that translocates an effector protein of streptococcal origin into the cytoplasm of a host cell. At least two proteins participate in CMT, the pore-forming molecule streptolysin O (SLO) and an effector protein with the characteristics of a signal transduction protein, the Streptococcus pyogenes NAD-glycohydrolase (SPN). In order to begin to elucidate the molecular details of the translocation process, we examined whether perfringolysin O (PFO), a pore-forming protein related to SLO, could substitute for SLO in the translocation of SPN. When expressed by S. pyogenes, PFO, like SLO, had the ability to form functional pores in keratinocyte membranes. However, unlike SLO, PFO was not competent for translocation of SPN across the host cell membrane. Thus, pore formation by itself was not sufficient to promote CMT, suggesting that an additional feature of SLO was required. This conclusion was supported by the construction of a series of mutations in SLO that uncoupled pore formation and competence for CMT. These mutations defined a domain in SLO that was dispensable for pore formation, but was essential for CMT. However, introduction of this domain into PFO did not render PFO competent for CMT, implying that an additional domain of SLO is also critical for translocation. Taken together, these data indicate that SLO plays an active role in the translocation process that extends beyond that of a passive pore.