We studied the differences among clinical isolates of Penicillin-resistant Streptococcus pneumoniae by polymerase chain reaction (PCR) of pbp2b gene, followed by serotyping and pulsed field gel electrophoresis (PFGE) analysis. Clinical isolates were recovered from sputum samples from patients with respiratory infection in Tottori University Hospital between June 1986 and May 1996. By PCR, altered pbp2b genes of the resistant isolates were detected in 76.5%. The percentages of the isolates that had altered pbp2b genes increased concomitantly with the minimum inhibitory concentration (MIC). By serotyping the percentage of 19F, 23F, 6B and 14 was 54.5%, 18.2%, 9.1% and 9.1% respectively. The frequency of isolates resistant to penicillin increased rapidly from 1991 in this hospital and most isolates belonged to serotype 19F. The resistant isolates in this hospital and 4 clinical resistant isolates of S. pneumoniae 19F in a second hospital were studied by PFGE. 14 of 18 resistant 19F isolates in this hospital and all 19F isolates from the second hospital presented an identical pattern. The remaining 4 samples were similar though not completely identical. These results indicate that the penicillin resistant 19F isolates have a common clonal origin and have spread rapidly from 1991 in this hospital.