RNA polymerase II (Pol II) transcription through nucleosomes is facilitated in vitro by the protein complex FACT (Facilitates Chromatin Transcription). Here we show that FACT is associated with actively transcribed Pol II genes on Drosophila polytene chromosomes. FACT displays kinetics of recruitment and of chromosome tracking in vivo similar to Pol II and elongation factors Spt5 and Spt6. Interestingly, FACT does not colocalize with Pol III-transcribed genes, which are known to undergo nucleosome transfer rather than disassembly in vitro. Our observations are consistent with FACT being restricted to transcription that involves nucleosome disassembly mechanisms.