Changes in vitamin D3 and its metabolites were investigated following UVB- and heat-treatment in the leaves of Solanum glaucophyllum Desf., Solanum lycopersicum L. and Capsicum annuum L. The analytical method used was a sensitive and selective liquid chromatography electrospray ionisation tandem mass spectrometry (LC–ESI-MS/MS) method including Diels–Alder derivatisation. Vitamin D3 and 25-hydroxy vitamin D3 were found in the leaves of all plants after UVB-treatment. S. glaucophyllum had the highest content, 200 ng vitamin D3/g dry weight and 31 ng 25-hydroxy vitamin D3/g dry weight, and was the only plant that also contained 1,25 dihydroxy vitamin D3 in both free (32 ng/g dry weight) and glycosylated form (17 ng/g dry weight).