A survey in 2000 to detect methicillin-resistant Staphylococcus aureus (MRSA) colonization in Vancouver downtown east side injection drug users (IDUs) revealed an MRSA nasal colonization incidence of 7.4%. This is a follow-up study to determine the current prevalence of MRSA colonization and to further characterize the isolates and risk factors for colonization. In this point prevalence study of MRSA nasal carriage among IDUs, nasal swabs were cultured to detect S. aureus. Isolates were studied for their antimicrobial susceptibility patterns and the presence of mecA and Panton-Valentine leukocidin (PVL) genes and by pulsed-field gel electrophoresis (PFGE). S. aureus was isolated from 119 of 301 (39.5%) samples; three (2.5%) participants had both methicillin-sensitive S. aureus (MSSA) and MRSA, resulting in 122 isolates. Of these, 54.1% were MSSA and 45.9% were MRSA, with an overall MRSA rate of 18.6%. USA-300 (CMRSA-10) accounted for 75% of all MRSA isolates; 25% were USA-500 (CMRSA-5). None of the USA-500 isolates were positive for PVL; 41 (97.6%) USA-300 isolates contained PVL. One MSSA isolate, from an individual also carrying USA-300, was positive for PVL. The PFGE pattern of this MSSA isolate was related to that of the MRSA strain. The antibiograms of USA-300 compared to USA-500 isolates showed 100% versus 7.1% susceptibility to trimethoprim-sulfamethoxazole (TMP-SMX) and 54.8% versus 7.1% susceptibility to clindamycin. MRSA nasal colonization in this population has increased significantly within the last 6 years, with USA-300 replacing the previous strain. Most of these strains are PVL positive, and all are susceptible to TMP-SMX.