Nosocomial Candida albicans infections have become a major cause of morbidity and mortality in neonates in neonatal intensive care units (NICUs). To determine the possible modes of acquisition of C. albicans in hospitalized neonates, we conducted a prospective study at Grady Memorial Hospital, Atlanta, Ga. Clinical samples for fungal surveillance cultures were obtained at birth from infants (mouth, umbilicus, and groin) and their mothers (mouth and vagina) and were obtained from infants weekly until they were discharged. All infants were culture negative for C. albicans at birth. Six infants acquired C. albicans during their NICU stay. Thirty-four (53%) of 64 mothers were C. albicans positive (positive at the mouth, n = 26; positive at the vagina, n = 18; positive at both sites, n = 10) at the time of the infant's delivery. A total of 49 C. albicans isolates were analyzed by restriction endonuclease analysis and restriction fragment length polymorphism analysis by using genomic blots hybridized with the CARE-2 probe. Of the mothers positive for C. albicans, 3 of 10 were colonized with identical strains at two different body sites, whereas 7 of 10 harbored nonidentical strains at the two different body sites. Four of six infants who acquired C. albicans colonization in the NICU had C. albicans-positive mothers; specimens from all mother-infant pairs had different restriction endonuclease and CARE-2 hybridization profiles. One C. albicans-colonized infant developed candidemia; the colonizing and infecting strains had identical banding patterns. Our study indicates that nonperinatal nosocomial transmission of C. albicans is the predominant mode of acquisition by neonates in NICUs at this hospital; mothers may be colonized with multiple strains of C. albicans simultaneously; colonizing C. albicans strains can cause invasive disease in neonates; and molecular biology-based techniques are necessary to determine the epidemiologic relatedness of maternal and infant C. albicans isolates and to facilitate determination of the mode of transmission.