Monoclonal antibodies (mAb's) have been raised against proteins in preparations of Z-discs isolated from honeybee fibrillar flight muscle. These antibodies have identified four Z-disc antigens on immunoblots of honeybee fibrillar proteins. Antibody alpha binds to the 90-100 kD protein, alpha-actinin; mAb P interacts with the protein, projectin, an extremely large polypeptide (greater than 600kD) found in the connecting filaments which link thick filaments to the Z-band in insect asynchronous flight muscle. Two other mAb's recognize previously uncharacterized insect Z-band proteins. Monoclonal antibody Z(400) binds to a pair of proteins with molecular masses near 400 kD and 600 kD. Antibody Z(175) recognizes two components, 158 kD and 175 kD, that are not only immunologically similar but have nearly identical peptide maps. Indirect immunofluorescence microscopy studies show that the proteins recognized by mAb's alpha, Z(175) and Z(400) are located at the Z-band, while the mAb P antigen is found on either side of it. Three of the four antibodies we have obtained recognize leg muscle proteins. Monoclonal antibodies alpha and P comigrate on SDS gels with analogous components from flight muscle. Only the smaller of the two proteins identified in flight muscle by mAb Z(400) is found in leg muscle, however. Furthermore, no Z(175) antigens have been detected in the non-fibrillar tissue by either monoclonal or polyclonal antibodies. Immunofluorescence microscopy studies localize the alpha and Z(400) antigens at the Z-line in leg muscle fibrils. Surprisingly, however, mAb P binds within the A-bands of synchronous fibres, not between the A- and Z-bands as in asynchronous fibrillar muscle.