Challenge of Dermacentor variabilis by hemocoel injection with Borrelia burgdorferi but not Bacillus subtilis or Escherichia coli provoked secretion of two low molecular weight peptides into the hemolymph plasma; the lower band co-migrated with a band previously identified as varisin (a tick defensin). These findings are consistent with reports that D. variabilis controls B. burgdorferi but not B. subtilis or E. coli by defensin-dependent bacteriolysis. Challenge of the tick midgut by capillary artificial feeding with bacteria also provoked expression of multiple low molecular weight peptides. In this case, however, all three bacteria elicited the response. Two bands, including the defensin-like peptide were expressed following challenge with B. subtilis and E. coli, but only the upper band following challenge with B. burgdorferi. Although they appeared intact, these spirochetes were no longer viable suggesting that borreliae in the midgut are controlled by a different method than the lytic response of the D. variabilis hemolymph. DD-RT-PCR revealed multiple mRNAs in the midgut of D. variabilis following challenge with B. burgdorferi, E. coli and Rickettsia montana. Although their identification remains to be determined, the large number of genes expressed in response to bacterial challenge presents intriguing possibilities for explaining the ability of the tick midgut to destroy invading microbes at the cellular level.