The axenic amastigotes of an Indian strain of L. donovani have been generated from the promastigote form at 37 degrees C in RPMI-1640 medium, pH 6.0 +/- 0.5, supplemented with 10 per cent heat inactivated foetal calf serum and these are being maintained through serial subculturing under the same conditions. The present study was carried out to differentiate axenic amastigote from the promastigote stage on the basis of their antigenic constitution and also to look for any immunoreactive antigen(s) specific to axenic amastigotes. SDS-PAGE analysis revealed a few stage specific and some conserved antigenic fractions in both the stages. On immunoblotting with immune sera raised against the membrane fractions of the axenic amastigotes, the 200 kDa antigenic fraction of axenic amastigote was found to be highly reactive. When the immune sera raised against the membranes of both stages were checked by immunofluorescence no cross reactivity was observed at higher dilutions. These findings showed that there are some antigenic diversities as well as similarities between the two stages of L. donovani cultured in vitro. Also, the 200 kD fraction of axenic amastigote appeared to be an immunodominant antigen specific to that stage.