Host functions involved in multiplication of single-stranded DNA phages S13, .vphi.R and .vphi.Kh-1 were examined using various DNA replication mutants of Escherichia coli. When the phage-resistant bacteria were to be tested, glycine spheroplasts or Ca2+-treated cells were transfected by the phage DNA. Replication of S13 and .vphi.R required host dnaB, dnaC(D), dnaE, dnaG and dnaZ gene products. The growth of .vphi.Kh-1 was dependent on cellular dnaE, dnaF(nrdA), dnaG and dnaZ functions, but not on dnaB and dnaC(D) activities. Multiplication of these phages absolutely relied on host rep function. Based on these host factor requirements, relationship among single-stranded DNA phages was discussed. Host range and antigenic properties of .vphi.Kh-1 were compared with those of the related phages.