Fifteen pediatric patients as well as the five nursing staff of the Burn Unit of the Hospital D. Estefania in Lisbon, Portugal, were assayed at weekly intervals over a five-month period in order to identify the nature and number of methicillin-resistant Staphylococcus aureus (MRSA) clones associated with colonization and wound infection. Methicillin resistance was confirmed by a mec-specific DNA probe. MRSA isolates were classified into chromosomal types (clones) on the basis of a variety of techniques: (i) ribotyping; (ii) restriction digestion by the endonuclease ClaI followed by Southern hybridization with the mecA-specific DNA probe and (iii) by hybridization with Tn554; and (iv) pulsed-field electrophoresis (PFE) of SmaI digests followed by (v) Southern hybridization with the mecA DNA probe. A sixth, physiological technique (population analysis) was used to define the mode of phenotypic expression of methicillin resistance in each isolate. All isolates carried a single, common polymorph (ClaI type III) of the mecA gene. Hybridization with Tn554 resolved these isolates to two novel patterns (alpha and beta), of which one (Tn554 alpha) was predominant (90%). This pattern could be further resolved to four closely related PFE types (A through D). In contrast, all isolates with the Tn554 beta pattern belonged to an additional, grossly different PFE type E. The Tn554 beta class was also unique in that these bacteria carried the mecA gene in a SmaI fragment smaller (about 170 kb) than that found in the alpha type strains (194 kb). Most isolates (83%) showed a single heterogeneous (population analysis Class 3) mode of resistance expression. The data demonstrate the full capacity of the globally rare (ClaI type III) MRSA clone for colonization and virulence. The results also document the stability of the complex heterogeneous resistance phenotype as well as the stability of the chromosomal types under conditions of in vivo carriage over a period of several months. In a few isolates the same mecA polymorph was present in several, grossly different genetic backgrounds, suggesting horizontal transfer of the mecA gene.