Abstract. Keratinocytes are the main cell population in the epidermis, where they coexist with a variety of other cell types. Their successful isolation and cultivation have afforded opportunities to study epidermal functions. Human keratinocytes have been studied most extensively, but their source is limited by the skin supply. In a previous work, we developed an in vitro co-culture model of porcine keratinocytes with porcine sensory neurites to investigate functional interaction. However, a detailed description of the isolation of porcine keratinocytes and their culture conditions has not been given in detail. Here, we present the isolation procedure and a characterization of keratinocytes derived from new-born piglets, using simple assays based on conventional and fluorescence microscopy. Media, coating substrates and plating densities were tested with respect to cell viability, proliferation and morphology. Growing keratinocytes in EpiLife keratinocyte growth medium (EKGM) on human collagen type I substrate was best to support proliferation. The minimum plated density was 500 viable cells/cm 2 for primary and 1000 viable cells/cm 2 for subcultured cells. Population doubling (PD) and generation time (tg) depended on the plating densities. Keratinocytes seeded at a density of 5000 viable cells/cm 2 had a PD of 4.36 ± 0.60 per passage and tg of 1.69 ± 0.24 days. Our results show that the optimal isolation and culture conditions for keratinocytes from piglets differ from those for keratinocytes from adult pigs and humans. Thus, the information obtained from the characterization allowed the performance and optimization of a co-culture and contributes to further investigations in epidermal homeostasis and cutaneous sensation.