A cell-free mRNA decay assay has been adapted to permit the kinetics of 3′ → 5′ exoribonuclease activities to be monitored in real time. RNA probes containing 5′ caps and 3′ poly(A) tails generated by transcription in vitro are 3′ labeled using fluorescein-N6-ATP and poly(A) polymerase. Release of fluorescein-conjugated adenosine residues from the 3′ end of the RNA substrate is monitored by a time-dependent decrease in fluorescence anisotropy in the presence of cytosolic proteins. To demonstrate the utility of the assay, an RNA probe was constructed containing a fragment of the c-myc 3′ untranslated region and an 85-base poly(A) tail. Following 3′ fluorescein labeling, the rate of 3′-terminal adenosine excision was monitored in the presence of an S100 cytosolic extract prepared from K562 erythroleukemia cells. Removal of the fluorescein-tagged A residues resolved to a first-order decay function, allowing the rate constant and enzyme-specific activity to be determined in this extract. Further applications and advantages of this technology are discussed.