A standard system for infecting ticks with the Lyme disease spirochete Borrelia burgdorferi Johnson, Schmid, Hyde, Steigerwalt & Brenner is defined. Rodents infected via tick feeding or inoculation of tick homogenates were more infectious to ticks than rodents infected with culture-derived spirochetes. White laboratory mice were more infectious than hamsters. Three strains of B. burgdorferi (JD1, B31, and WI210) produced batches of infected ticks with > 80% infection when mice were infected with tick-derived material. Ixodes dammini Spielman, Clifford, Piesman & Corwin were 3.6x more efficient than I. pacificus Cooley & Kohls in acquiring and maintaining infection with two California strains of B. burgdorferi, originally isolated from I. pacificus.