Laminin-5-rich extracellular matrix derived from 804G cells (804G-ECM) induces spreading, improves glucose-stimulated insulin secretion, and increases survival and proliferation of rat pancreatic beta-cells. The aim of the study was to determine growth signaling pathways activated by ECM with a particular focus on Ca²⁺-dependent transcription factors. 804G-ECM increased rat beta-cell proliferation and this stimulation was glucose- and Ca²⁺-dependent. NF-B nuclear translocation as well as IkB gene expression were also Ca²⁺-dependent. Inhibition of NF-B almost completely blocked 804G-ECM-stimulated beta-cell proliferation as did the soluble IL-1 receptor antagonist IL-1Ra. 804G-ECM-induced proliferation was also blocked by cyclosporin A and the VIVIT peptide suggesting involvement of NFAT/calcineurin. Use of selective inhibitors further implicated other pathways in this process. Inhibition of PI3 kinase and protein kinase A both prevented beta-cell replication stimulated by 804G-ECM. Conversely, inhibition of MAPK, JNK, p38 and GSK3 increased beta-cell proliferation on 804G-ECM. Our results suggest that Ca²⁺ entry which is necessary for increased beta-cell proliferation on 804G-ECM is also involved in 804G-ECM induced NF-B activity. It is proposed that increased cytosolic Ca²⁺ leads to activation of the transcription factors NFAT and NF-B that in turn increase beta-cell proliferation. Activation of PI3 kinase by 804G-ECM also increases proliferation possibly by synergistic co-activation of NFAT via inhibition of GSK3, while IL-1 may amplify the process by feed-forward activation of NF-B. Conversely, inhibition of the MAPK pathway increased beta-cell proliferation indicating a counter-regulatory restraining role for this signaling pathway.