The agar-cloning technique of Blastocystis hominis has been observed in both solid and semisolid agar using Iscove's modified Dulbecco's medium. In this study, Philippine isolates of B. hominis were grown by pour-plate method in semisolid agar using Locke's solution. Inoculated plates contained 0.7% agar, 10% horse serum, and 0.1% sodium thioglycollate. Plates were incubated at 37 degrees C in a microaerophilic jar for 7-10 days. Biconvex disk-shaped colonies were seen abound at the bottom half of the medium. Colonies growing at the agar-glass interface were flat and consisted of thin layers of cells. From these colonies, large amoeboid cells were frequently seen on the periphery, whereas smaller cells were concentrated at the core. Analysis of the SSU rDNA genetically established the identity of the clones to be B. hominis. This is the first report on agar cloning of Blastocystis in a compound medium.