Streptococcus pneumoniae cell wall and pneumolysin are important contributors to pneumococcal pathogenicity in some animal models. To further explore these factors in middle ear inflammation caused by pneumococci, penicillin-induced inflammatory acceleration was studied by using three closely related pneumococcal strains: a wild-type 3 strain (WT3), its pneumolysin-negative derivative (P-1), and its autolysin-negative derivative (A-1). Both middle ears of chinchillas were inoculated with one of the three pneumococcal strains. During the first 12 h, all three strains grew in vivo at the same rate, and all three strains induced similar inflammatory cell responses in middle ear fluid (MEF). Procaine penicillin G was given at 12 h to one-half of the animals in each group, and all treated chinchillas had sterile MEF at 24 h. Penicillin significantly accelerated MEF inflammatory cell influx into WT3- and P-1-infected ears at 18 and 24 h in comparison with the rate for penicillin-treated A-1-infected ears. Inflammatory cell influx was slightly, but not significantly, greater after treatment of WT3 infection than after treatment of P-1 infection. Interleukin (IL)-1β and IL-6, but not IL-8, concentrations in MEF at 24 h reflected the penicillin effect on MEF inflammatory cells; however, differences between treatment groups were not significant. Results suggest that pneumococcal otitis media pathogenesis is triggered principally by the inflammatory effects of intact and lyric cell wall products in the middle ear, with at most a modest additional pneumolysin effect. Investigation strategies that limit the release of these products or neutralize them warrant further investigation.