New multisubstrate-type inhibitors of the deoxynucleoside kinases have been synthesized, tested for their specificity as soluble inhibitors of enzymes from Lactobacillus acidophilus, and used to construct media for affinity chromatography. Each inhibitor was a deoxynucleoside 5'-adenosine 5"'-P1,P4-tetraphosphate (abbreviated dNp4A, where dN represents a dAdo, dCyd, dGuo, or dThd moiety linked through its 5'-hydroxyl to the terminal phosphate of adenosine tetraphosphate). At micromolar concentrations, each inhibitor strongly and specifically inhibited the corresponding deoxynucleoside kinase. Each of the four Lactobacillus deoxynucleoside kinase activities was selectively retained on its homologous dNp4A-Sepharose affinity medium. The activity was eluted on addition of the respective dNp4A with up to 70% recovery and 300-500-fold purification (relative to an ammonium sulfate fraction). Whereas dThd kinase was retained only by the dTp4A column, a portion of the dAdo kinase activity was retained, along with all the dCyd kinase or dGuo kinase, on dCp4A- or dGp4A-Sepharose, respectively, and coeluted with these activities. Conversely, all three activities were quantitatively retained on dAp4A-Sepharose, without competition from either dCyd or dGuo, and were eluted simultaneously upon addition of dAp4A. These observations further confirm the understanding that this organism employs paired, and presumably bifunctional, kinases, namely dCyd/dAdo kinase and dGuo/dAdo kinase, along with a separate thymidine kinase.