Islet cell antigen 69 (ICA69), previously implicated as an autoantigen in autoimmune insulin-dependent diabetes mellitus (IDDM), was produced using baculovirus-mediated expression in Spodopterafrugiperda (Sf9) insect cells. In these cells the protein was effectively expressed and ICA69 carrying C-terminal histidine-hexapeptide could be efficiently purified using immobilized metal chelate affinity chromatography. Screening of patient and control sera using this protein as an antigen in time-resolved fluoroimmunoassay (TR-FIA) identified 4/50 of patients with IDDM and 6/73 of patients with rheumatoid arthritis (RA) to be positive for ICA69 antibodies. The number of positives did not differ significantly between patients and control subjects but the level of binding was higher in sera from RA patients compared to that of control sera (P = 0.003). The results show that some subjects have specific autoreactive antibodies against the ICA69 protein produced with recombinant technology.