The temporal variation in the abundance and proportion of highly respiration-active bacteria in the eutrophic lakes Esrum and Frederiksborg Slotssø was determined with the redox dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC). In addition, a comparative late summer study was undertaken across a gradient of nutrient enrichment in Danish lakes. The purpose was to investigate the importance of substrate (chlorophyll) and temperature for the control of CTC-active cells (CTC+). The abundance of CTC+ cells was much lower and more variable than the total number of cells counted after 4',6-diamidino-2-phenylindole (DAPI) staining. The proportion of CTC+ cells in Lake Esrum and Frederiksborg Slotssø was normally <5%, and between 2.5 and 20% in 14 other lakes. The abundance as well as the proportion of CTC+ cells increased with chlorophyll in Lake Esrum and Frederiksborg Slotssø, and chlorophyll explained 43% of the variability in CTC+ abundance. In the comparative study, the abundance of CTC+ cells increased along the chlorophyll gradient, which explained 49% of the variability. The results showed that the abundance and, to a lesser degree, the proportion of CTC+ bacteria were controlled by substrate supply. One consequence of the low abundance of active bacteria is that in situ growth rates scaled to CTC+ cells are 3- to 7-fold higher than those scaled to DAPI counts. It is suggested that studies on factors controlling bacterioplankton activity at the single-cell level should be investigated scaled to active cells.