Isothiazole dioxides have been shown to inhibit Trypanosoma brucei protein farnesyltransferase (PFTase) in isolated enzyme, but elicited only a minor effect on mammalian PFTase. In the present study we have evaluated the effect of 3-diethylamino-4-(4-methoxyphenyl)-isothiazole 1,1-dioxides with different substituents at C5, on rat PFTase and protein geranylgeranyltransferase-I (PGGTase-I) with the final aims to improve the potency against mammalian PFTase and to identify new compounds with antiproliferative properties. For these purposes, in vitro and cell culture models have been utilized. The results showed that isothiazole dioxides with C4-C5 double bond and sulfaryl substituted at the C5 position but none of the dihydro-derivatives, were able to inhibit in vitro PFTase in a concentration dependent manner (IC50 ranging from 8.56 to 1015 microM). Among those, compound 6n (C5; methyl-S) displayed 500-fold higher inhibitory potency on PFTase than PGGTase-I. Compound 6n was shown to affect rat smooth muscle cell (SMC) proliferation at concentrations similar (IC50 = 61.4 microM) to those required to inhibit [3H]-farnesol incorporation into cellular proteins (-44.1% at 100 microM). Finally, compound 6n interferes with rat SMC proliferation by blocking the progression of G0/G1 phase without inducing apoptosis, as assessed by [3H]-thymidine incorporation assay and flow cytometry analysis. Taken together, we described a new PFTase inhibitor containing the isothiazole dioxide moiety that affects mammalian protein farnesylation and SMC proliferation by inhibiting G0/G1 phase of the cell cycle.