Repeated bottleneck passages of RNA viruses result in accumulation of mutations and fitness decrease. Here, we show that clones of foot-and-mouth disease virus (FMDV) subjected to bottleneck passages, in the form of plaque-to-plaque transfers in BHK-21 cells, increased the thermosensitivity of the viral clones. By constructing infectious FMDV clones, we have identified the amino acid substitution M54I in capsid protein VP1 as one of the lesions associated with thermosensitivity. M54I affects processing of precursor P1, as evidenced by decreased production of VP1 and accumulation of VP1 precursor proteins. The defect is enhanced at high temperatures. Residue M54 of VP1 is exposed on the virion surface, and it is close to the B-C loop where an antigenic site of FMDV is located. M54 is not in direct contact with the VP1-VP3 cleavage site, according to the three-dimensional structure of FMDV particles. Models to account for the effect of M54 in processing of the FMDV polyprotein are proposed. In addition to revealing a distance effect in polyprotein processing, these results underline the importance of pursuing at the biochemical level the biological defects that arise when viruses are subjected to multiple bottleneck events.