The major inducible 70-kDa heat shock protein (hsp72) increases measles virus (MV) transcription and genome replication. This stimulatory effect is attributed to hsp72 interaction with two highly conserved hydrophobic domains in the nucleocapsid protein (N) C terminus of Edmonston MV. These domains are known as Box-2 and Box-3. A single amino acid substitution in Box-3 of Edmonston MV (i.e., N522D) disrupts hsp72 binding. The prevalence of the N522D substitution in contemporary wild-type MV isolates suggests that this sequence has been positively selected. The present work determined if the N522D substitution enhances viral fitness and the degree to which any fitness advantage is influenced by hsp72 levels. Both parent Edmonston MV (Ed N) and an N522D substitution mutant (Ed N-522D) exhibited similar growth on Vero and murine neuroblastoma cells and in cotton rat lung, although Ed N-522D virus exhibited an attenuated in vitro response to hsp72 overexpression. In contrast, mixed infections showed a significantly reduced in vitro and in vivo fitness of Ed N-522D virus. Results support the involvement of additional selectional pressures that maintain the circulation of virus containing N-522D despite the cost to viral fitness.