Abstract Oligonucleotides labeled with a single fluorophore (fluorescein or tetramethylrhodamine) have been used previously as fluorogenic substrates for a number of DNA modifying enzymes. Here, it is shown that such molecules can be used as fluorogenic probes to detect the template-dependent binding of deoxynucleotide triphosphates by DNA polymerases. Two polymerases were used in this work: the Klenow fragment of the Escherichia coli DNA polymerase I and the Bacillus stearothermophilus polymerase, Bst. When complexes of these polymerases with dye-labeled hairpin-type oligonucleotides were mixed with various deoxynucleotide triphosphates in the presence of Sr2+ as the divalent metal cation, the formation of ternary DNA–polymerase–dNTP complexes was detected by concentration-dependent changes in the fluorescence intensities of the dyes. Fluorescein- and tetramethylrhodamine-labeled probes of identical sequences responded differently to the two polymerases. With Bst polymerase, the fluorescence intensities of all probes increased with the next correct dNTP; with Klenow polymerase, tetramethylrhodamine-labeled probes increased their fluorescence, but the intensity of fluorescein-labeled probes decreased on formation of ternary complexes with the correct incoming nucleotides. The use of Sr2+ as the divalent metal ion allowed the formation of catalytically inactive ternary complexes and obviated the need for using 2′,3′-dideoxy-terminated oligonucleotides as would have been needed in the case of Mg2+ as the metal ion.