Borrelia burgdorferi exists in nature via an enzootic cycle whereby the organism must adapt to the diverse environmental conditions provided inside the arthropod transmission vector and the mammalian reservoir hosts. B. burgdorferi genes shown to be regulated by temperature, pH and/or cell density during the organism's growth in culture medium were assayed for expression during various stages of the tick feeding cycle by reverse transcription-polymerase chain reaction (RT-PCR). ospA, ospC, flaB, erpA/I/N, erpB/J/O, rev and mlpA, were transcriptionally active following the larval and nymphal stages of feeding as determined by qualitative RT-PCR. During tick resting periods between feedings, ospC, mlpA and rev transcription were undetectable, in contrast to ospA, flaB, erpA/I/N and erpB/J/O. bba64, a gene induced by environmental changes in culture and expressed during mammalian infection, was not detectable during any of the tick life cycle phases. Quantitative PCR to determine B. burgdorferi genome equivalents in these tick samples using DNA co-purified with the RNA allowed an estimation of gene expression relative to the numbers of B. burgdorferi present in the ticks. Although the spirochete totals varied widely between individual tick pools of fed, replete nymphs, the relative expression ratios between individual target genes following a nymphal feed were comparable. Similarly, borrelial gene transcription from the larval feeding and the nymphal feeding were observed and compared. These findings analogize B. burgdorferi gene expression observed by environmental stimuli in vitro with the transcriptional activity occurring during the organism's infectious cycle within the tick.