The infection dynamics of the tick-transmitted organism Ehrlichia chaffeensis were investigated in white-tailed deer (Odocoileus virginianus) using different routes of inoculation. Six deer were each inoculated with 5.4×10⁶ DH82 cells infected with E. chaffeensis (Arkansas strain) by three different routes: intravenous (n=2), subcutaneous (n=2), and intradermal (n=2). Two control deer were inoculated with uninfected cells. Infections were monitored for 54 days and were continued in one deer from each E. chaffeensis inoculated group for an additional 31 days. All deer inoculated with E. chaffeensis seroconverted (≥1: 64) and became 16S rDNA polymerase chain reaction and/or cell culture positive by post-inoculation day 15. There was no apparent difference in susceptibility to infection between deer inoculated by different routes for the first 50 days based on detection of E. chaffeensis infection by PCR assay of blood or culture isolation. These results demonstrate infection of deer by intradermal and subcutaneous routes for the first time.