It would have been helpful if you also posted your code and/or graph, because now we have to *assume* the things you did... Anyway, if I plot/overlay my gene expression data using pathview, rectangles correspond indeed to genes/enzymes. If you plot fold changes, red is 'increased', and green is 'decreased'. If in a pathway some genes/rectangles are red, and others are green, this thus means that these genes are oppositely regulated. What this regarding pathway activity /biological perspective is up to interpretation from your side and might not be straight-forward; for example if an inhibitor is decreased this may result in increased activity.... When you have a rectangle that has multiple colors (example here) this usually means you have overlaid data form multiple experiments (e.g. time points, treatments) in the same plot (but again without your code it is impossible to say). The legend values always go from -1 to 1, because these are the default settings. Please note that in pathview the color-coded values are original and not standardized. The color key gives the range, values beyond that range are converted to the closest extreme, i.e. values>1 converted to 1, and values< -1 converted to -1. Please check arguments limit, low, mid, high in pathview documentation.