Differential gene expression by Borrelia burgdorferi spirochetes during mammalian infection facilitates their dissemination as well as immune evasion. Modulation of gene transcription in response to host immunity has been documented with the outer surface protein C, but the influence of transcription of other genes is largely unknown. A low-density array (LDA) was developed to study transcriptional activity of 43 B. burgdorferi genes and 19 host genes that may be involved in various host-agent interactions. Gene transcription in heart, joint, and muscle tissue was compared in immunocompetent C3H and immunodeficient C3H-scid mice during early (3 weeks) and late (2 months) B. burgdorferi infection. Among all tissue types, levels of relative transcription of over 80% of B. burgdorferi genes tested were one- to nine-fold less in C3H mice compared to C3H-scid mice. At the later time point, all genes were transcribed in C3H-scid mice, whereas transcription of 16 genes out of 43 tested was not detected in analyzed tissues of C3H mice. Our data suggest that during infection of immunocompetent mice, a majority of B. burgdorferi genes tested are downregulated in response to acquired host immunity. LDA revealed variable patterns of host gene expression in different tissues and at different intervals in infected mice. Higher levels of relative expression for IL-10 during both early and late infection were detected in heart base, and it was unchanged in the tibiotarsal joint. Comparative analysis of B. burgdorferi and host genes transcriptional activity revealed that increased flaB mRNA during early infection was followed by increases of CCL7, CCL8, interleukin-10 (IL-10), and tumor necrosis factor-α (TNF-α) in all assessed tissue types. LDA represents a valuable approach for sensitive and quantitative gene transcription profiling and for understanding Lyme borreliosis.