The presence of oogonia in the ovaries of adult females is typical in species with a broadcast spawning reproductive strategy, including invertebrates and lower vertebrates. In sea urchins, difficulties in the study of oogonia arise from the small number of these cells and the lack of specific markers for their identification. Therefore, more reliable methods are needed for identifying and manipulating oogonial cells in quantities sufficient for experimentation. Homologs of the DEAD-box RNA helicase vasa expressed in germline cells have been proposed for use as markers to detect germline cells in diverse species. We have developed a method for the isolation of sea urchin oogonia by using immunocytochemistry with vasa antibodies, together with reverse transcription and the polymerase chain reaction to detect the expression of Sp-vasa and Sp-nanos2 homologs and a morphological approach to identify germline cells in sea urchin ovaries and cell fractions isolated from the ovarian germinal epithelium. This method has allowed us to obtain 15%-18% of small oogonia with 70%-75% purity from the total amount of isolated germ cells. Our findings represent the first methodological basis for obtaining cell populations containing sea urchin oogonia; this method might be useful as a tool for further investigations of the early stages of sea urchin oogenesis.