As part of viral replication, the HIV-1 reverse transcriptase (HIV-RT) makes a DNA copy of the RNA genome of the virus. It is a mutagenic polymerase, which leads to the rapid development of resistance in patients being treated with antiviral drugs. To study the mechanism by which this enzyme makes frequent errors, we have developed and used a set of variably sized nonpolar uridine analogues (rH, rF, rL, rB) in RNAs to probe its functional steric flexibility in nucleotide incorporation. Our kinetics studies show that the enzyme pairs all the analogues selectively with adenine, with a preference for dichloro analogues. However, unlike high-fidelity replicative DNA polymerases, this reverse transcriptase shows little sensitivity to size increases beyond the optimum, which gives evidence for unusually high steric flexibility. This suggests that at least part of the mutagenic behavior of the HIV-1 virus is caused by active site flexibility in its replicating enzyme.