Background: Network motifs provided a " conceptual tool" for understanding the functional principles of biological networks, but such motifs have primarily been used to consider static network structures. Static networks, however, cannot be used to reveal time- and region-specific traits of biological systems. To overcome this limitation, we proposed the concept of a " spatiotemporal network motif," a spatiotemporal sequence of network motifs of sub-networks which are active only at specific time points and body parts.Results: On the basis of this concept, we analyzed the developmental gene regulatory network of the Drosophila melanogaster embryo. We identified spatiotemporal network motifs and investigated their distribution pattern in time and space. As a result, we found how key developmental processes are temporally and spatially regulated by the gene network. In particular, we found that nested feedback loops appeared frequently throughout the entire developmental process. From mathematical simulations, we found that mutual inhibition in the nested feedback loops contributes to the formation of spatial expression patterns.Conclusions: Taken together, the proposed concept and the simulations can be used to unravel the design principle of developmental gene regulatory networks. © 2012 Kim et al.; licensee BioMed Central Ltd.