Antigen-specific CD4+ T lymphocyte responses contribute to protective immunity against Babesia bovis, however the antigens that induce these responses remain largely unknown. A proteomic approach was used to identify novel B. bovis antigens recognized by memory CD4+ T cells from immune cattle. Fractions obtained from merozoites separated by continuous-flow electrophoresis (CFE) that contained proteins ranging from 20 to 83 kDa were previously shown to stimulate memory CD4+ lymphocyte responses in B. bovis-immune cattle. Expression library screening with rabbit antiserum raised against an immunostimulatory CFE fraction identified a clone encoding a predicted 78 kDa protein. BLAST analysis revealed sequence identity of this B. bovis protein with Plasmodium falciparum fatty acyl coenzyme A synthetase (ACS) family members (PfACS1-PfACS11), and the protein was designated B. bovis acyl-CoA synthetase 1 (ACS1). Southern blot analysis indicated that B. bovis ACS1 is encoded by a single gene, although BLAST analysis of the preliminary B. bovis genome sequence identified two additional family members, ACS2 and ACS3. Peripheral blood lymphocytes and CD4+ T cell lines from B. bovis-immune cattle proliferated significantly against recombinant ACS1 protein, consistent with its predicted involvement in protective immunity. However, immune sera from cattle recovered from B. bovis infection did not react with ACS1, indicating that epitopes may be conformationally dependent.