Prenylation is a post-translational modification of proteins that involves the attachment of an isoprenoid group derived from mevalonic acid, either 15-carbon farnesyl or 20-carbon geranylgeranyl, to a specific carboxy-terminal domain of acceptor proteins. Three prenyl transferase enzymes have been identified so far. In this paper we report the presence of two prenyl transferases in the HaCaT human keratinocyte cell line. Chromatography of a cytosolic extract from these cells resolved a farnesyl protein transferase (FPT) and geranylgeranyl protein transferase-I (GGPT-I) whose activities were measured using a novel peptide-based assay. Both enzymes were inhibited dose dependently by zaragozic acids A and C. Zaragozic acid C was more active towards the FPT than GGPT-I while zaragozic acid A inhibited both enzymes with similar potency. Incubation of HaCaT cell homogenates with [3H] prenyl precursors resulted in the labelling of a number of proteins which was increased when the cells were pretreated with an inhibitor of hydroxymethylglutaryl CoA reductase. Given the role of prenylated proteins in proliferative and inflammatory processes, our finding that prenyl transferases capable of prenylating endogenous substrates are also present in keratinocytes suggests that these enzymes might provide novel therapeutic targets of dermatological importance.