It is now clear that in addition to activating several complex kinase pathways (Smad, MAP kinase, PI3 kinase), TGFβ also acts by elevating [Ca2+] within the cytosol of human pulmonary fibroblasts. Ca2+/calmodulin-dependent protein kinase II (CamK II) is also known to regulate gene expression in fibroblasts. In this study, we examined the interactions between calcium signaling, activation of CamK and other kinases, and extracellular matrix (ECM) gene expression. Human pulmonary fibroblasts were cultured and stimulated with artificially-generated Ca2+-pulses in the absence of TGFβ, or with TGFβ (1 nM) or vehicle in the presence of various blockers of Ca2+ signaling. PCR and Western blotting were used to measure gene expression and protein levels, respectively. We found that Ca2+-pulses in the absence of TGFβ increased ECM gene expression in a pulse frequency-dependent manner, and that blocking Ca2+ signaling and the CamK II pathway significantly reduced TGFβ-mediated ECM gene expression, without having any effects on other kinase pathways (Smad, PI3 kinase or MAP kinase). We also found that TGFβ elevated the expression of CamK IIβ and CamK II, while siRNA silencing of those two subtypes significantly reduced TGFβ-mediated expression of collagen A1 and Fibronectin 1. Our data suggest that TGFβ induces the expression of CamK IIβ and CamK II, which in turn are activated by TGFβ-evoked Ca2+ waves in a frequency-dependent manner, leading to increased expression of ECM proteins.