Pneumococci with decreased susceptibility or resistant to penicillin (PRP) have been isolated with an increasing frequency in France. Among PRP, isolates of serotypes 23F and 9V were the most frequently recovered in our children's hospital. Penicillin-resistance is due to the appearance of altered penicillin binding proteins (PBPs) with reduced affinity for beta-lactam antibiotics. 3 PBPs have been well studied, 2b, 2x and 1a, and the sequences of their genes have been determined. Our molecular epidemiological study of 14 PRP 9V and 26 PRP 23F isolated mainly from otitis in 1993-94, consisted of determining chromosomic restriction patterns (Apa I) by pulsed-field gel electrophoresis, and restriction patterns (Hinf I) of PBP genes pbp 2b, pbp 2x and pbp 1a after PCR. All the PRP 9V exhibited the same pulsotype and identical patterns for each of the genes pbp 2b, pbp 2x and pbp 1a, suggesting a clonal origin. The origins of PRP 23F were more heterogenous: 5 clones could be defined, with one predominant clone composed of 20 isolates. Most of the PRP 23F shared identical profiles for the genes pbp 2b, pbp 2x and pbp 1a with the PRP 9V, suggesting a horizontal transfer of DNA. Molecular markers, which provide more informations than serotyping, were useful to clarify the complex epidemiology of PRP.