During myofibril formation, Z‐bodies, small complexes of alpha‐actinin and associated proteins, grow in size, fuse and align to produce Z‐bands. To determine if there were changes in protein dynamics during the assembly process, Fluorescence Recovery after Photobleaching was used to measure the exchange of Z‐body and Z‐band proteins with cytoplasmic pools in cultures of quail myotubes. Myotubes were transfected with plasmids encoding Yellow, Green, or Cyan Fluorescent Protein linked to the Z‐band proteins: actin, alpha‐actinin, cypher, FATZ, myotilin, and telethonin. Each Z‐band protein showed a characteristic recovery rate and mobility. All except telethonin were localized in both Z‐bodies and Z‐bands. Proteins that were present both early in development in Z‐bodies and later in Z‐bands had faster exchange rates in Z‐bodies. These results suggest that during myofibrillogenesis, molecular interactions develop between the Z‐band proteins that decrease their mobility and increase the stability of the Z‐bands. A truncated construct of alpha‐actinin, which localized in Z‐bands in myotubes and exhibited a very low rate of exchange, led to disruption of myofibrils, suggesting the importance of dynamic, intact alpha‐actinin molecules for the formation and maintenance of Z‐bands. Our experiments reveal the Z‐band to be a much more dynamic structure than its appearance in electron micrographs of cross‐striated muscle cells might suggest. Cell Motil. Cytoskeleton 61:34–48, 2005. © 2005 Wiley‐Liss, Inc.