In an attempt to develop a technique of fluorescence in situ hybridization (FISH) to detect DNA in Paramecium, we examined three different DNA probes, total genomic DNA, genomic DNA encoding C5 phagosomal membrane antigen, and telomere, prepared from P. multimicronucleatum. In accordance with the conventional method, total genomic DNA probe was denatured at 75–80°C for 2 min, and the cells were denatured at 75, 80, 85, or 90°C for 5 or 10 min. The homogeneous hybridization signal with the total genomic DNA probe was obtained at 85°C for 10 min, or at 90°C for 5 min or 10 min. This condition was applied for the smaller DNA probe, C5 (1. 3 kb, the size close to detection limits), in which the expected tiny signals throughout the macronuclear nucleoplasm was observed. However, the condition was not successful for the telomeric DNA probe. The hybridization signals of telomeric DNA were only detected when both cells and probes were denatured simultaneously in a same denaturation buffer. In the case of the simultaneously denatured samples, the preservation of the nuclear morphology was relatively poor, however, the signals of the telomeric DNA probe were observed in the periphery of the macronucleus. As a negative control, an irrelevant 40 kb human cosmid probe was examined by both conventional and simultaneous denaturation methods, and none of the hybridization signal was observed with this probe. These results suggest that the current methods allow us to follow localization of the specific sequences within the macronuclear compartment.