A method to identify the earliest stages (initials) of Pleurotus pulmonarius and Coprinus cinereus fruit body formation was developed. Light microscopy and Cryo-SEM were used to confirm that even the smallest structures to take up stain were initials of fruit bodies. An approach combining histological staining (flooding the Petri dish with 1% toluidine blue in 1% boric acid (w/v) for 15 min) and image analysis allowed the number of fruit bodies formed on Petri dishes to be quantified easily. Use of the vital stain Janus green (0·001% aqueous, w/v) allowed continued observation of living tissue so that the proportion of fruit bodies that matured (30%) could be established. The method was also effective on wheat straw cultures and could be used to monitor development of mature fruit bodies. It is a promising tool in the study of physiological processes involved in fruit body initiation.