The surface-associated molecules of the second (L2), third (L3) and fourth (L4) larval stages of Dirofilaria immitis were characterized employing radiolabeling techniques and SDS-PAGE analysis. Major labeled components of 35 kDa and 6 kDa were present in extracts from IODO-GEN-labeled L2 and L3 parasites. The results of lactoperoxidase-catalyzed reactions also demonstrated that L2 and L3 stages of D. immitis have a similar repertoire of surface-associated antigens. However, in contrast to the results obtained with IODO-GEN, lactoperoxidase reactions labeled components with apparent molecular weights of 66, 48, 25, 16.5 and 12 kDa. The similarities in the molecular weights of the L2 and L3 surface-associated components and the results of immunoprecipitation experiments which demonstrated that antibodies produced against the 35 kDa molecule from D. immitis L3s also recognize the 35 kDa component from L2 parasites suggest that synthesis of the molecules found at the surface of mature infective larvae begins as early as day 6 of development in the mosquito, D. immitis L4s emerged from the molting process with a repertoire of surface-associated antigens distinct from those found on L2s and L3s. IODO-GEN labeling of D. immitis L4s showed major surface-associated molecules with apparent molecular weights of 57, 40, 25, 12 and 10 kDa when analyzed under non-reducing conditions. In addition to molecules of 57, 40, 25, 12 and 10 kDa, extracts of D. immitis L4s labeled with lactoperoxidase contained additional major bands at 45, 43 and 8 kDa. Metabolic labeling experiments demonstrated a shift in the amount and complexity of the excretory/secretory products released by D. immitis during L3 to L4 development.