From June to July 1998, two episodes of Candida tropicalis fungemia occurred in the Aristotle University neonatal intensive care unit (ICU). To investigate this uncommon event, a prospective study of fungal colonization and infection was conducted. From December 1998 to December 1999, surveillance cultures of the oral cavities and perinea of the 593 of the 781 neonates admitted to the neonatal ICU who were expected to stay for >7 days were performed. Potential environmental reservoirs and possible risk factors for acquisition of C. tropicalis were searched for. Molecular epidemiologic studies by two methods of restriction fragment length polymorphism analysis and two methods of random amplified polymorphic DNA analysis were performed. Seventy-two neonates were colonized by yeasts (12.1%), of which 30 were colonized by Candida albicans , 17 were colonized by C. tropicalis , and 5 were colonized by Candida parapsilosis . From December 1998 to December 1999, 10 cases of fungemia occurred; 6 were due to C. parapsilosis , 2 were due to C. tropicalis , 1 was due to Candida glabrata , and 1 was due to Trichosporon asahii (12.8/1,000 admissions). Fungemia occurred more frequently in colonized than in noncolonized neonates ( P < 0.0001). Genetic analysis of 11 colonization isolates and the two late blood isolates of C. tropicalis demonstrated two genotypes. One blood isolate and nine colonization isolates belonged to a single type. The fungemia/colonization ratio of C. parapsilosis (3/5) was greater than that of C. tropicalis (2/17, P = 0.05), other non- C. albicans Candida spp. (1/11, P = 0.02), or C. albican s (0/27, P = 0.05). Extensive environmental cultures revealed no common source of C. tropicalis or C. parapsilosis . There was neither prophylactic use of azoles nor other risk factors found for acquisition of C. tropicalis except for total parenteral nutrition. A substantial risk of colonization by non- C. albicans Candida spp. in the neonatal ICU may lead to a preponderance of C. tropicalis as a significant cause of neonatal fungemia.