Free-living infective larvae Strongyloides venezuelensis were cultured in Dulbecco's modified Eagle's medium at 25 and 37 °C, and development to the parasitic stage was evaluated using morphological, protein and antigenicity criteria. Few larvae cultured at 25 °C showed development whereas, in most of the larvae cultured at 37 °C, there appeared characteristic changes such as a bulb-like head and droplets under the cuticle with an increase of body width of the larvae. The results obtained from two-dimensional polyacrylamide gel electrophoresis(2D–PAGE) revealed that the protein spot patterns of the larvae cultured at 25 and 37 °C were differentiated by 17 specific spots. In addition, Western blot analysis combined with 2D–PAGE for reaction with serum obtained from an infected rat revealed that protein spots showing immunodominant antigen at 37 °C were almost the same as those of the larvae recovered from the rats rather than those of the larvae at 25 °C. These results strongly suggested that a temperature shift from 25 to 37 °C has an important role in the development of free-living infective larvae to the parasitic stage of S. venezuelensis. The culture system established in the present study was useful for biological and biochemical studies in the development from/of the free-living to the parasitic stage of Strongyloides species.