Borrelia burgdorferi gene expression within the guts of engorging Ixodes scapularis ticks was examined by use of differential immunoscreening and differential expression with a customized amplified library. Fourteen chromosomal genes involved in energy metabolism, substrate transport, and signal transduction and 10 (4 chromosomal and 6 plasmid) genes encoding putative lipoproteins and periplasmic proteins were preferentially expressed in engorging ticks. These data demonstrate a new approach to the global analysis of B. burgdorferi genes that are preferentially expressed within the vector during feeding.