The reliability of vaginal cytology for determining the optimal mating period is controversial. The importance of the site for obtaining the smear (the vestibule or the vagina) and the best staining method are also unclear. Therefore, we investigated the reliability of vaginal cytology as compared to plasma progesterone concentrations for determining the optimal mating period. Additionally, the influence of different locations for obtaining the smears and of the staining methods, i.e. May-Grünwald-Giemsa or Papanicolaou, a method that stains keratin in superficial cells, was examined. In total, the vaginal cytology of 35 bitches during the follicular phase was examined three times a week, until the optimal time for mating was established, based on plasma progesterone concentrations measured with a 125Iodine radio-immunoassay. The results indicate that vaginal cytology is not useful for determining the optimal mating period in bitches, since mating according to cytological findings would have resulted in only 28% of the bitches being mated at the proper time. The site for obtaining the smear and the staining method did not affect accuracy. It is therefore concluded that cytology is not useful in breeding management to determine the optimal mating period. It can be used, however, to determine the stage of the cycle in general, e.g. the early part of the follicular phase or the metoestrus and the detection of disorders in the follicular phase. For this purpose, a smear from the vestibule, stained with a morphological staining method, provides sufficient information.