Many B. burgdorferi genes are regulated at the level of transcription during B. burgdorferi passage from ticks to mammals. Particular spirochete outer surface proteins of interest are OspA, OspC, and vlsE. The messenger RNA (mRNA) levels produced by these three genes were determined by a quantitative reverse transcription PCR (q-RT-PCR) procedure for spirochete populations in nymphal I. scapularis midguts and salivary glands at specific intervals during the feeding process. The mRNA values were compared to that of a standard, the mRNA levels of the constitutively expressed Flagellin (fla) gene. The levels of OspA and vlsE did not increase markedly in the midgut during feeding, but the mRNA levels of OspC increased significantly during feeding. In tick salivary glands, OspA mRNA levels actually decreased during feeding, while OspC levels increased six orders of magnitude. The mRNA levels of vlsE in tick salivary glands increased significantly only during the last 2 days of tick feeding. Overall, OspA mRNA was more abundant in tick midguts, whereas OspC and vlsE mRNA was more abundant in tick salivary glands. Further studies on the regulation of B. burgdorferi transcription activity during the act of transmission will lead to a better understanding of spirochete transmission dynamics, and hopefully facilitate the development of novel ways of interrupting the spread of Lyme disease.