Chlamydia trachomatis serovar L2 is an obligate intracellular bacterium which is internalized in target epithelial cells by endocytosis and resides within a membrane-bound vesicle. Over the next several hours following entry, individual serovar L2-containing vesicles fuse with one another to form a single membrane-bound vesicle (or inclusion) within which the microcolony develops. The experiments reported here directly examined the pH of vesicles containing chlamydiae. The pH was determined by measuring emission ratios of the fluorescent, pH-sensitive probe SNAFL (5-[and 6-]-carboxyseminaphthofluorescein-1, succinimidyl ester) conjugated to chlamydiae. The pH remained above 6.0 at 2, 4, and 12 h after infection, while the pH of vesicles contained heat-killed organisms fell 5.3. In the presence of amines, which raise the pH of acidic compartments, C. trachomatis inclusion formation was unaffected. Inactivation of Na+,K+ -ATPases, the ion pumps responsible for maintaining a pH above 6 within early endocytic vesicles, inhibited the growth of C. trachomatis within epithelial cells. Preventing vesicular acidification by inhibiting the vacuolar proton ATPase did not affect chlamydial growth. Thus, chlamydiae do not reside within highly acidic vesicles and avoid the pathway leading to lysosomes.