Vascular smooth muscle cells (VSMCs) have the innate ability to modulate cellular phenotype in response to environmental factors such as vessel damage or disease. This phenotypic plasticity ranges between a contractile phenotype, the most common form found in vessels, to a synthetic phenotype which are unable to contract though play a vital role in vessel remodelling. Variations in VSMC phenotype are evident with changes in cell morphology, expression of SMC markers, proliferation and migratory characteristics. In vitro culture methods utilising calf serum and enzymatic digestion are known factors which can shift contractile VSMCs to a more synthetic phenotype. If assessing cellular contraction, changes in phenotype in vitro may lead to findings which may have little or no in vivo relevance. Numerous in vitro culture techniques have been suggested to maintain VSMCs in a contractile phenotype; however, no comparative study has been conducted using human aortic smooth muscle cells. In this study, VSMC gene marker expression was utilised to compare culture methods which may induce a contractile phenotype. Aortic smooth muscle cells (Lonza, Slough, UK) were cultured between passages 7 and 11 in basal medium (DMEM supplemented with 10% FCS), and compared to cells grown in DMEM with 5% FCS or Lonza medium (EBM+SmGM-2 BulletKit). In addition, cells were cultured in basal medium on plates coated with either fibrillar or monomeric collagen, or treated with heparin. Expression of mRNA for smoothelin, a contractile phenotype marker, was up-regulated at all passages in cells cultured on monomeric collagen, compared to basal medium. Expression of mRNAs for other contractile markers: caldesmon, heavy chain 11 smooth muscle and actin alpha 2 smooth muscle, was also favourable in monomeric collagen coated cultures. Cells cultured on fibrillar collagen also followed a similar pattern of mRNA expression to monomeric, though to a lesser extent. VSMCs grown in Lonza medium and DMEM supplemented with 5% FCS exhibited mRNA marker expression suggestive of a more synthetic phenotype, compared to basal medium. Further to this, VSMCs treated with heparin showed favourable mRNA expression of contractile markers, which in cases was better than that observed with monomeric collagen; however, given that heparin can effect cellular contraction, this may not be as viable a method to induce a contractile phenotype. Taken together, these findings suggest that culturing VSMCs in Lonza medium may promote the more proliferative synthetic phenotype and may serve to build up cell populations. These cells could subsequently be transferred onto cultureware coated with monomeric collagen which would induce a contractile phenotype and thus be appropriate for in vitro studies of cellular contraction.