Premise of the Study:Datura stramonium is a pharmacologically and evolutionarily important plant species in the family Solanaceae. Stable transformation methodology of this species would be advantageous for future genetic studies. Methods:In vitro plant regeneration and Agrobacterium tumefaciens-mediated transformation techniques were developed for D. stramonium based on methods reported for tomato. A binary vector containing pAtUBQ10::erGFP was used for transformation. Results:We recovered primary transformants harboring the green fluorescent protein (GFP) transgene that resulted in expression of fluorescence in all tissues analyzed. Transformants were allowed to self-pollinate, and two of five progeny contained the GFP transgene and displayed fluorescence identical to the primary transformants. Discussion:We have demonstrated the first stable transformation in the genus Datura. This is a key first step to study the genetic basis of traits in this evolutionarily interesting species