A fluorimetric assay for measuring terminal deoxynucleotidyl transferase activity in purified and crude enzyme preparations has been developed. Etheno-substituted deoxynucleotides are shown to be substrates of the enzyme. The assay involves polymerization of the fluorescent nucleotide 1,N6-ethenodeoxyadenosine triphosphate (epsilon dATP) on an oligodeoxynucleotide initiator, [poly(deoxyadenylic acid) with an average chain length of 50 residues] under the reaction conditions used in the standard radiometric assay. The incorporation of epsilon dATP into polymer is quantitated by fluorescence after isolation and nuclease digestion of the product. The enzymological properties of etheno substrates were also determined. Epsilon dATP binds about twofold tighter than dATP to terminal transferase, but has a twofold-lower catalytic rate. However etheno substitution does not affect initiator binding. The fluorimetric assay is suitable for clinical analysis of terminal transferase in human leukemias, and may be a useful adjunct to recently developed immunochemical methods which detect protein, not activity.