Graph theory provides a useful and powerful tool for the analysis of cellular signaling networks. Intracellular components such as cytoplasmic signaling proteins, transcription factors, and genes are connected by links, representing various types of chemical interactions that result in functional consequences. However, these graphs lack important information regarding the spatial distribution of cellular components. The ability of two cellular components to interact depends not only on their mutual chemical affinity but also on colocalization to the same subcellular region. Localization of components is often used as a regulatory mechanism to achieve specific effects in response to different receptor signals. Here we describe an approach for incorporating spatial distribution into graphs and for the development of mixed graphs where links are specified by mutual chemical affinity as well as colocalization. We suggest that such mixed graphs will provide more accurate descriptions of functional cellular networks and their regulatory capabilities and aid in the development of large-scale predictive models of cellular behavior.