For determination of the oestrous cycle in rats classical Papanicolaou technique has long been used successfully. Instead of using many stains in Papanicolaou, staining the vaginal secretions with only methylene blue has also been defined. Recently a new technique in which vaginal samples are directly examined under light microscope has been introduced. The aim of this study was to assess the reliability of this new technique by comparing it with the classical staining techniques. From 20 Wistar rats 60 vaginal samples were collected with a micropipette, three from each. Briefly, the vagina was flushed two to three times then the fluid was placed onto a glass slide. The fluid was equally distributed onto three glass slides. The glass slides were coded. Two samples were stained with Papanicoloau and methylene blue while the other one was examined directly. Determination of the phases of the oestrous cycle was made by the same histologist who was blinded to the groups and coding system. After determination of the oestrous phase in all samples, the results were compared and it was found that the results were matching. In conclusion, the same results can be obtained with the direct examination technique and this technique is reliable, so there is no need to use relatively time-consuming, less practical and more expensive techniques such as Papanicolaou or methylene blue.