Recent work suggests that some native conformations of proteins can vary with temperature. To obtain an atomic-level description of this structural and conformational variation, we have performed all-atom, explicit-solvent molecular dynamics simulations of bovine pancreatic ribonuclease A (RNase A) up to its melting temperature (Tm ≈ 337 K). RNase A has a thermal pretransition near 320 K [Stelea, S.D, Pancoska, P., Benight, A.S., Keiderling, T.A. (2001) Prot. Sci. 10, 970—978]. Our simulations identify a conformational change that coincides with this pretransition. Between 310 and 320 K, there is a small but significant decrease in the number of native contacts, β-sheet hydrogen bonding, and deviation of backbone conformation from the starting structure, and an increase in nonnative contacts. Native contacts are lost in β-sheet regions and in α1, partially due to movement of α1 away from the β-sheet core. At 330 and 340 K, a nonnative helical segment forms at residues 15–20, corresponding to a helix observed in the N-terminal domain-swapped dimer [Liu Y.S., Hart, P.J., Schulnegger, M.P., Eisenberg, D. (1998) Proc. Natl. Acad. Sci. USA, 95, 3437—3432]. The conformations observed at the higher temperatures possess native-like topology and overall conformation, with many native contacts, but they have a disrupted active site. We propose that these conformations may represent the native state at elevated temperature, or the N′ state. These simulations show that subtle, functionally important changes in protein conformation can occur below the Tm.