A novel, infectious Borrelia burgdorferi that expresses green fluorescent protein (GFP) was developed to examine the utility of this marker protein to label live bacteria during infection processes. Use of a borrelial erpAB promoter to direct gfp transcription supported previous indications that B. burgdorferi expresses erp genes during chronic mammalian infection and during acquisition by feeding ticks. Live bacteria fluoresced and were seen to be located extracellularly in infected mice and within midguts of infected ticks. These results indicate that transcriptional fusions between B. burgdorferi promoters and gfp can be useful tools to examine spirochete gene expression in vivo.