Borrelia burgdorferi has over 20 circular and linear plasmids that may be involved with infecting mice and ticks. Nine spirochete strains with varying plasmid profiles were used to identify plasmids necessary for nymphal tick infection. All spirochete strains tested infected ticks guts, but to different degrees. The plasmids lp5, lp28-1 and cp9 were not required for infecting tick guts, while loss of lp25 and lp28-4 was associated with reduced gut infectivity. A reduction in the ability of spirochetes to invade salivary glands was seen in bacteria that didn’t have lp28-1, whereas cp9 was not required. Linear plasmid 25 was studied in more detail due to its requirement for establishing infection in mice and ticks. Strains missing lp25 are able to infect mice if the bbe22 gene on lp25 is provided on a vector. A infection cycle was mimicked by feeding larvae on mice injected with the parental, lp25^(-), or lp25^(-) bbe22-complemented spirochete strains. Ticks were also artificially infected to study tick infections independent of mouse infections. Spirochetes missing lp25 were impaired in their ability to infect ticks. When a lp25^(-) strain was complemented with bbe22, the ability to infect ticks was partially restored. Complementation with bbe22 allowed spirochetes lacking lp25 to establish short term infections in ticks, but in most cases the infection prevalence was lower than the wild type strain. In addition, the number of infected ticks decreased over time, suggesting that another gene(s) on lp25 is required for persistence in ticks and completion of a infection cycle.