This paper reports on the application of surface chemical gradients to study mammalian cell interactions with synthetic surfaces and investigates if the cell response on certain parts of the gradient is the same as that on uniform surfaces of equivalent chemistry. The gradients, formed using a diffusion-controlled plasma polymerisation technique, were fabricated such that cell response to a large range of different chemistries on a single sample could be investigated. Surface chemical gradients from hydrophobic plasma polymerised hexane (ppHex) to a more hydrophilic plasma polymerised allylamine (ppAAm), previously used to control cell density within 3D tissue-engineering scaffolds, were formed on glass coverslips. Surface characterisation was carried out to determine water contact angles (WCA), elemental composition, coating thickness and topography of the chemical gradients. Cell response was assessed following culture of 3T3 fibroblasts on both steep and shallow gradients. Fibroblasts adhered and proliferated preferentially on ppAAm (WCA approximately 60 degrees ) showing a gradual decreasing cell density towards the hydrophobic ppHex (WCA approximately 93 degrees ). Experiments on a uniform ppAAm surface revealed that there was a significant difference in cell density when compared to the gradient samples. The initial number of cells that adhered to the surface was confirmed as the difference between the uniform and graduated ppAAm samples, and it is assumed that this difference relates to different cell-cell signalling processes and/or greater protein production from surrounding cells on these two samples formats.