Nontypeable Haemophilus influenzae (NTHi) and Moraxella catarrhalis are common causative agents of human mucosal infections. To formulate a mucosal vaccine against these pathogens, recombinant lipidated P4 (rLP4) and P6 (rLP6) proteins of NTHi and ubiquitous cell surface protein A (UspA) of M. catarrhalis were used for active immunization experiments in a mouse nasal challenge model. BALB/c mice were immunized intranasally with these proteins formulated with a chemically synthesized adjuvant, RC529 in an aqueous formulation (RC529-AF). Three weeks after the last immunization, these animals were challenged intranasally with NTHi strain SR7332.P1 and nasal colonization measured 3 days later. To determine local and systemic immune responses, bronchoalveolar washes (BAW) and sera were collected prior to NTHi challenge. The serum and mucosal samples were analyzed by ELISA for rLP4, rLP6 and UspA2 protein-specific IgG, IgG subclass and IgA antibody titers and bactericidal titers were determined against the TTA24 and 430-345 strains of M. catarrhalis. Results of these experiments show that these proteins combined with RC529-AF administered intranasally to mice elicited (1) significantly increased rLP4/rLP6/UspA2 protein-specific circulating IgG and IgA antibody responses; (2) local rLP4/rLP6/UspA2-specific IgA responses in the respiratory tract; and (3) more than a two log reduction of nasal colonization of NTHi strain SR7332 from the nasal tissues of mice. The serum IgG subclass distribution was predominantly IgG2a, representing a Th1 response. The antiserum also exhibited bactericidal activities to several strains of M. catarrhalis. These data indicate that intranasal immunization with rLP4/rLP6/UspA2 proteins combined with RC529-AF may be able to provide a way for inducing local mucosal immunity and for prevention of otitis media in children.