Fine‐resolution measurements of phytoplankton and physical parameters were made from 31 May to 14 June 2005 in the Ría de Pontevedra (Spain), which is subject to seasonal upwelling. The main objective of this work was to elucidate physical‐biological interactions leading to subsurface aggregations of toxin‐producing microalgae (Pseudo‐nitzschia spp. and Dinophysis spp.). A sequence of upwelling‐relaxation‐upwelling‐downwelling events was recorded with a moored Acoustic Doppler Current Profiler (ADCP). Thin layers (TLs) of Pseudo‐nitzschia spp. and other diatoms (up to 30 µg chlorophyll a L−1) developed and persisted in the pycnocline above cooler (12.5ºC) upwelled water but were vertically displaced and even dispersed during downwelling. The establishment of steep pycnoclines after upwelling pulses and the formation of TLs of Pseudo‐nitzschia spp. and other diatoms suggest that pycnoclines may act as retention areas for these populations. Their vertical displacement during downwelling would explain different patterns observed in the contamination of benthic resources and raft‐mussels. A decimeter‐scale segregation of Pseudo‐nitzschia micans and Dinophysis acuminata populations was observed. The population of D. acuminata, present since March 2005 in Ría de Pontevedra, was never found within the pycnocline, did not perform any significant vertical migration, and was not dispersed during upwelling. Instead, it formed patches (up to 8 × 103 cell L−1) in the warmer (15‐18ºC) surface (0‐4 m) waters associated with a diurnal thermocline, and it spread throughout the ría into a near‐surface layer during relaxation and downwelling. These results demonstrate the importance of considering species‐specific behavior to predict the location of cell maxima.