In the last 30 years, 81 Streptococcus thermophilus bacteriophage isolates were collected from industrial yogurt (n = 40) and cheese (n = 41) fermentation. Forty-six distinct restriction patterns of phage DNA (11 in yogurt and 35 in cheese) were observed. The phages were investigated for host range, serological properties, and DNA homology to study whether these three independent techniques can be used to classify the phages into taxonomic groups. Yogurt factory-derived phages were classified into the same two subgroups by serology, host range analysis, and hybridization with subgroup-specific DNA sequences. Cheese factory-derived phages, however, could not be classified: the 35 cheese phage isolates with distinct restriction patterns showed 34 different host ranges. All but one cheese phage isolate showed serological cross-reactivity with yogurt phages. A phage DNA fragment that hybridized with all phage DNA samples was cloned, establishing the genetic relatedness of all S. thermophilus phages from our collection. With the sequence information from an unusually conserved S. thermophilus phage DNA element (H. Brüssow, A. Probst, M. Frémont, and J. Sidoti, Virology 200:854-857, 1994), a PCR-based phage detection method was developed for cheese whey from a factory that produced mozzarella cheese with complex undefined starter mixes. PCR allowed the detection of phages in cheese whey (detection limit, 10(3) PFU/ml) which could not be detected by dot blot hybridization techniques (detection limit, 10(7) PFU/ml).