Microphytobenthos (MPB) contribute significantly to primary production in many estuarine and coastal waters. Although the vertical migration of microphytobenthos is well integrated into benthic studies of intertidal areas, the presence and importance of such migration has not yet been investigated in subtidal areas. In situ measurements and sampling in the Bay of Brest, French Atlantic coast, in November 2003 and May, July and September 2004 and subsequent laboratory experiments showed that pulse amplitude modulated (PAM) fluorometry can be employed to follow vertical migration of subtidal MPB. Steady-state fluorescence (F) was highly correlated with chlorophyll a (chl a) concentrations in the top 200 µm of sediments and could be used to detect in situ changes in MPB biomass. When (as in the present study) subtidal variations in light and temperature are negligible compared to those in intertidal areas, variations in fluorescence parameters due to changes in chl a concentrations are much greater than those due to photochemical and non-photochemical quenching. In situ surveys during 3 seasons showed a daily bell-like periodicity in F, regardless of tidal oscillations. Sediment cores incubated at constant light and temperature displayed similar day/night fluorescence variations, indicating an endogenous rhythm, and that the diel cycle is the main factor triggering migration. Our results revealed a difference in the functioning of subtidal and intertidal MPB, in which migration is linked not only to diel but also to tidal cycles. In future studies of subtidal benthic primary production, care should be taken in sampling strategies, particularly in selecting sampling times, as the migratory behaviour of the MPB may greatly alter estimates of available or actively photosynthesising biomass.