Topical application of pantothenate is widely used in clinical practice for wound healing. Previous studies identified a positive effect of pantothenate on migration and proliferation of cultured fibroblasts. However, these studies were mainly descriptive with no molecular data supporting a possible model of its action. In this study, we first established conditions for an in vitro model of pantothenate wound healing and then analysed the molecular effects of pantothenate. To test the functional effect of pantothenate on dermal fibroblasts, cells were cultured and in vitro proliferation tests were performed using a standardized scratch test procedure. For all three donors analysed, a strong stimulatory effect of pantothenate at a concentration of 20 microg/ml on the proliferation of cultivated dermal fibroblasts was observed. To study the molecular mechanisms resulting in the proliferative effect of pantothenate, gene expression was analysed in dermal fibroblasts cultivated with 20 microg/ml of pantothenate compared with untreated cells using the GeneChip Human Exon 1.0 ST Array. A number of significantly regulated genes were identified including genes coding for interleukin (IL)-6, IL-8, Id1, HMOX-1, HspB7, CYP1B1 and MARCH-II. Regulation of these genes was subsequently verified by quantitative real-time polymerase chain reaction analysis. Induction of HMOX-1 expression by pantothenol and pantothenic acid in dermal cells was confirmed on the protein level using immunoblots. Functional studies revealed the enhanced suppression of free radical formation in skin fibroblasts cultured with panthenol. In conclusion, these studies provided new insight in the molecular mechanisms linked to the stimulatory effect of pantothenate and panthenol on the proliferation of dermal fibroblasts.