The fraction of planktonic bacteria in oligo- and mesotrophic lakes that can be classified by in situ hybridization has been significantly increased with an optimized protocol. By the use of white polycarbonate filters, CY3-monolabeled oligonucleotide probes, and high quality fluorescence filter sets between 29 and 64% of the cells visualized by 4',6-diamidino-2-phenylindole (DAPI) can be detected with a bacterial probe or group-specific probes by epifluorescence microscopy without further digital image enhancement.