Nosocomial neonatal candidiasis is a major problem in infants requiring intensive therapy. The subjects of this retrospective study were nine preterm infants admitted to the neonatal intensive care unit of the Hospital Central de Asturias between March 1993 and August 1994. The infants were infected with or colonized by Candida albicans. Five patients developed C. albicans bloodstream infections. A total of 36 isolates (including isolates from catheters and parenteral nutrition) were examined for molecular relatedness by PCR fingerprinting and restriction fragment length polymorphism (RFLP) analysis. The core sequence of phage M13 was used as a single primer in the PCR-based fingerprinting procedure, and RFLP analysis was performed with C. albicans-specific DNA probe 27A. Both techniques were evaluated with a panel of eight C. albicans reference strains, and each technique showed eight different patterns. With the 36 isolates from neonates, each technique enabled us to identify by PCR and RFLP analysis seven and six different patterns, respectively. The combination of these two methods (composite DNA type) identified eight different profiles. A strain with one of these profiles was present in three patients and in their respective catheters. Patients infected with or colonized by this isolate profile were clustered in time. Among the other patients, each patient was infected over time and at multiple anatomic sites with a C. albicans strain with a distinct DNA type. We conclude that C. albicans was most commonly producing long-term colonizations, although horizontal transmission probably due to catheters also occurred.