The role of heterotrophic nanoflagellates (HNAN, size 2-20 μm) in grazing on bacteria and mineralization of organic matter in stratified Lake Vechten was studied.Quantitative effects of manipulation and fixation on HNAN were checked. Considerable losses were caused by centrifugation, even at low speed, and by filtration if the vacuum exceeded 3 kPa. HNAN were well preserved for several weeks with unbuffered 5% formaldehyde as well as with 0.3 to 1% glutaraldehyde (final concentrations). However, the chlorophyll autofluorescence of phototrophic nanoflagellates was strongly decreased by formaldehyde, whereas it could be preserved for a few days with 1% glutaraldehyde. Loss of autofluorescence and of cells was prevented for at least one year if prepared primulin-stained slides were stored in a freezer.During summer stratification in 1985, HNAN showed peak densities in the microaerobic lower metalimnion at 6-7 m depth. In this stratum, high HNAN numbers (more than 30 x 10 6HNAN.l -1) coincided with high bacterial production, as estimated by thymidine incorporation. Despite the high production, bacterial numbers (about 5 x 10 9bacteria.l -1) did not peak in the metalimnion but were similar throughout the whole water column, indicating that the produced bacteria were consumed by HNAN. A steep decline of HNAN was followed by a peak of ciliates. Thus, HNAN seemed to be a link in the food web between bacteria and ciliates. Batch incubations with selectively filtered lake water, with bacteria alone- and with bacteria plus protozoa, indicated ingestion rates of about 7 bacteria.HNAN -1.h -1, and suggested that the HNAN are able to balance the estimated bacterial population doubling times of about 1 day.The breakdown of organic carbon (C) of dead cyanobacterial cell walls added to selectively filtered lake water was not accelerated by protozoa. However, HNAN and ciliates strongly increased the mineralization of nitrogen (N) and especially phosphorus (P), both from Synechococcus cell walls and cell extract. Bacterial growth ...