Information is limited on the factors that may affect the preputial differential epithelial cell count in healthy intact dogs. Our objectives were to establish RIs of the preputial differential epithelial cell count in dogs and investigate the potential impact of signalment, presence of gross preputial discharge or neutrophils, preputial cleaning, and normal estrogen variation on the differential cell count. We enrolled prospectively 50 client-owned, healthy dogs in our study. Preputial cytology smears were obtained, and we performed a 4-part (parabasal, intermediate, superficial, cornified) differential cell count, before and after gentle preputial cleaning. We found higher counts of fully cornified cells (median: 11-12%) compared to previous studies. The cleaning procedure significantly decreased the intermediate (p = 0.002) and increased the superficial (p = 0.006) cells. Age was positively associated with intermediate (p = 0.007) and negatively with superficial (p = 0.01) and cornified (p = 0.02) cells. The concentration of estrogens was negatively associated with the percentage of parabasal cells (p = 0.04). In healthy dogs, a variable impact may be expected in the preputial differential epithelial cell count, by the cleaning of the prepuce before sampling, the age of the dogs, and the normal variation of estrogen concentration.