Atherosclerosis is an inflammatory disease characterised by increased expression of adhesion molecules for leukocytes on both the surface of dysfunctional endothelium and on smooth muscle cells (SMC) within the lesion. It is also characterised by altered SMC phenotypic expression, indicated by a decreased volume fraction of myofilaments (V(v)myo) [1,2] and changes in gene expression [3]. The present study used an in vitro model to investigate, by immunofluorescence staining and flow cytometry, the influence of phenotype on vascular SMC expression of the adhesion molecule for leukocytes, intracellular adhesion molecule-1 (ICAM-1), and the regulatory mechanisms involved in this process. Smooth muscle cells with a high V(v)myo, freshly isolated from rat aortic media, expressed little or no ICAM-1 and this could not be induced by interleukin-1beta (IL-1beta). As SMC modulated phenotype, indicated by decreasing V(v)myo over the first 5 days of culture, there was a concomitant increase in ICAM-1 expression. At day 9 of primary culture, when SMC cultures had returned to the high V(v)myo phenotype, ICAM-1 expression was markedly lower. However, these cells retained the capacity to express ICAM-1 in response to IL-1beta. After several passages in culture, cells (with a low V(v)myo) constitutively expressed ICAM-1, with levels further up-regulated in response to IL-1beta. These changes in ICAM-1 expression were not related to proliferative state, since similar results were obtained with growth arrested SMC. Investigation of signalling pathways involved in regulating ICAM-1 expression by primary vascular SMC suggested a complex regulatory mechanism. Activation of adenyl cyclase (with forskolin) caused a significant increase in cells expressing ICAM-1. Treatment with inhibitors of protein kinase C (chelerythrine chloride), protein tyrosine kinase (genistein), or the transcription factor NF-kappaB (PDTC) had no significant effect on IL-1-induced ICAM-1 expression. However, in the presence of serum, both genistein and PDTC caused a significant increase in basal expression. The results indicate that ICAM-1 expression by SMC is phenotype-dependent, with expression evident only after cells have modulated to a low V(v)myo phenotype. They also indicate the existence of complex regulatory mechanisms, possibly involving the SMC cytoskeleton.