A float greenhouse transplant study was conducted at the Virginia Tech Southern Piedmont Center (Blackstone, Virginia, United States) to evaluate the interaction of water source and fertilizer analysis on tobacco transplant production. One water source (municipal) had a relatively low buffering capacity with a neutral pH and a relatively low total alkalinity. The second water had a high pH and alkalinity level plus a conductivity level twice that of the first water. Data were collected of bay water and soilless media pH, electrical conductivity, and nutrient content throughout the season. Samples for tissue analysis were collected just prior to first clipping and when seedlings were ready for transplanting. Both bay water and soilless medium pH were higher from all four fertilizers with the well water source. Trends in soilless medium pH corresponded to the acid/base reaction of the fertilizer treatments. Tissue analysis showed differences in a few nutrient elements but not of much consequence and no deficiency or toxicity symptoms were observed. Results of this study illustrate the impact of water source on greenhouse transplant production factors as well as the need to match fertilizer chemistry to water source chemistry.