More than 50% of the nontypeable (NT) pneumococcal strains received in our laboratory for reference purposes are isolated in sporadic cases of conjunctivitis. To determine the genetic structure of the population of these NT conjunctival strains, we analyzed 75 pneumococci (40 NT and 35 typeable) isolated from conjunctivas and 30 (15 NT and 15 typeable) isolated from other sources. The NT and typeable conjunctival strains grouped in separate clusters, whereas NT and typeable pneumococci isolated from other sources were similarly distributed. NT conjunctival strains belonged to two well-differentiated clonal lineages. The first, represented by three newly described sequence types, featured fully antibiotic susceptible strains and appeared to be characteristic of conjunctival tissue; the second, represented by the previously described ST344, had a pattern of multiresistance to penicillin, tetracycline, and erythromycin and shared a genetic background with some NT strains isolated from other sources.Streptococcus pneumoniae is an important pathogenic bacterium associated with pneumonia, septicemia, meningitis, and otitis. It is also a common cause of acute conjunctivitis, particularly in children, but also in adults (11,19). For reference purposes, our laboratory receives many pneumococcus samples from all origins that have been isolated in Spanish hospitals (8). Around one-third of the strains isolated from children below the age of 6 months that were studied in our laboratory between 1990 and 1999 caused acute conjunctivitis (9).Pneumococcal serotyping usually fails to detect a small number of strains that do not react with antipneumococcal typing sera. Nontypeable (NT) strains are infrequently isolated from sterile clinical specimens (2.2%), in which case they are rarely implicated as causes of invasive disease (2, 12), since they are otherwise relatively common in nonsterile samples (10%). The identification of these NT pneumococci is dubious (14, 17), particularly in nonsterile specimens, and they may be confused with other Streptococcus species. The association between the presence of NT isolates and the occurrence of conjunctivitis was first suggested in 1977 (10) in a retrospective study of the incidence of capsular types in a Boston hospital between 1935 and 1974.Further studies associated NT strains with outbreak and sporadic cases of conjunctivitis (1,6,18,20), and a recent report has confirmed NT S. pneumoniae-like strains isolated from an outbreak of epidemic conjunctivitis as being S. pneumoniae (3).In the last 10 years, approximately 50% of the NT pneumococcal strains received in our laboratory have been isolated from cases of conjunctivitis, and the frequency of these noncapsular strains was five times that found in other pathologies (laboratory data).In general, the NT strains have been characterized in cases related to outbreaks, but as yet we have little information concerning those strains isolated from sporadic cases. The purpose of this study was to characterize NT pneumococcal strains isolated...