During serial undiluted passage of a clonal population of foot-and-mouth disease virus (FMDV C-S8c1) in BHK-21 cells, two species of defective RNA were generated and selected. Sequence analysis revealed that they included deletions within the L-coding region, and retained the correct reading frame for viral protein synthesis. These deleted RNAs directed the synthesis of capsid protein VP1, were packaged in particles sedimenting with standard virus, required homologous infectious helper virus in order to produce viral particles, but did not interfere with the replication of helper virus. While detection of defective particles in FMDV required more than 100 serial passages, once produced, these defective RNAs could be stably maintained upon further passages in the FMDV C-S8c1 quasispecies. Furthermore, a high fitness, monoclonal-antibody-resistant virus was able to replace the standard virus and support the amplification of the deleted particles. This is the first description of naturally occurring, defective particles of FMDV.