We report the synthesis and characterization of 5-thioacetamidofluorescein-uridine 5'-triphosphate (5-SF-UTP), and its application to the characterization of the environment of the nascent RNA during transcription. This analog specifically replaced UTP as a transcription substrate for Escherichia coli and T7 RNA polymerases, and yeast RNA polymerase III. Escherichia coli transcription complexes containing analog incorporated at only position +21 of the RNA were prepared. The RNA was then elongated in the absence of analog, moving the fluorescent group further away from the enzyme active site, and the fluorescence polarization was measured. Analog positioned near the 3' end of the transcript exhibited significantly increased polarization relative to that of free probe, consistent with the constrained environment of the RNA in the DNA-RNA hybrid. Analog positioned 14 nucleotides from the 3' end exhibited significantly decreased polarization relative to that at the 3' end of the RNA, but only slightly above that of free RNA, suggesting that the probe was on the solvent-exposed surface of the polymerase. Molecular modeling of these analog-substituted RNAs produced structures consistent with the experimental data. The excellent substrate properties of this analog make it useful for the characterization of the environment of RNA not only during transcription and translation, but in any type of ribonucleoprotein complex.