A classic in vitro model of renal cyst and tubule formation utilizes the Madin-Darby canine kidney (MDCK) cell line, of which two strains exist. The vast majority of studies of cyst and tubule formation utilizing MDCK cells have been performed with MDCK Strain II cells. MDCK Strain II cells form hollow cysts in a three-dimensional collagen matrix over the course of ten days and tubulate in response to hepatocyte growth factor (HGF), which increases levels of active (phosphorylated) extracellular-signal regulated kinase (ERK) 1/2. Here we demonstrate that MDCK Strain I cells also form cysts when grown in a collagen matrix; however, MDCK Strain I cell cysts spontaneously initiate the primary steps in tubulogenesis. Analysis of time-lapse microscopy of both MDCK Strain I and Strain II cell cysts during the initial stages of tubulogenesis demonstrates a highly dynamic process with cellular extensions and retractions occurring rapidly and continuously. The ability of MDCK Strain I cell cysts to spontaneously initiate tubulogenesis is due to relatively higher levels of active ERK in MDCK Strain I, compared to Strain II, cells. The presence of either of two distinct inhibitors of ERK activation (U0126 and PD098059) prevents tubulogenesis from occurring spontaneously in MDCK Strain I cell cysts and, in response to HGF, in Strain II cell cysts. The difference between MDCK Strain I and Strain II cell lines is likely explained by differing embryologic origins, with Strain I cells being of collecting duct, and hence ureteric bud, origin. Ureteric bud cells also have high levels of active ERK, spontaneously tubulate in our in vitro collagen gel system, and tubulogenesis is inhibited by U0126 and PD098059. These results suggest that a seminal event in kidney development may be the activation of ERK in the mesonephric duct/ureteric bud cells that are destined to form the collecting tubules.