The preparation of morphologically distinct monolayers of cells by tissue culture of dissected seminiferous tubules and of the intertubular fraction of the adult rat testis is reported. The cells comprising the monolayers obtained by tissue culture of the intertubular fraction of the testis represent interstitial cells, identification being based on light microscopy, 3-β hydroxysteroid dehydrogenase histochemistry, electron microscopy and the ability of the cells to localize 125I-labelled lh.