The behavior of a double male strain of Escherichia coli K12 has been compared to that of its parents and a primary F' strain carrying F14 in order to determine whether the genome of the double male, responsible for the double origin mode of gene transmission, is composed of one or two linkage groups. F-prime plasmids carrying leu and pyrB and argG and metC have been detected following mating an F(-)recA(-) recipient with the double male. Their existence strongly supports the contention that the double male is composed of a single linkage group with two integrated sex factors. Data from acridine orange curing experiments places the frequency of double male cells in the population of growing cells in the two-chromosome configuration as less than one percent. Evidence for a unique origin and terminus of DNA replication deduced by the density labelling and transduction of double male DNA supports the contention that the double male is best considered a cell with a single chromosome carrying two integrated F plasmids.