A promoter is an essential structural component of a gene that controls its transcription activity in different development stages and in response to various environmental stimuli. Knowledge of promoter functionality in heterologous systems is important in the study of gene regulation and biotechnological application. In order to explore the activity of the pepper capsaicin synthase gene (PUN1) promoter, gene constructs of pPUN1::GUS (for β-glucuronidase) and pPUN1::NtKED (for a tobacco wound-responsive protein) were introduced into tobacco and tomato, respectively, and their activities were examined. Higher levels of GUS staining intensity and transcription were detected in ovary, anther and pollen than other tissues or organs in tobacco plants. Likewise, transgenic tomato fruits had a higher level of pPUN1::NtKED gene expression than the leaf and flower. The PUN1-driven gene expression can be transiently induced by wounding, heat (40 °C) and the capsaicinoid biosynthetic pathway precursor phenylalanine. When compared to the reported pPUN1::GUS-expressing Arabidopsis, the PUN1 promoter exhibited a more similar pattern of activities among pepper, tobacco and tomato, all Solanaceae plants. Our results suggest the potential utility of this tissue-preferential and inducible promoter in other non-pungent Solanaceae plants for research of gene function and regulation as well as in the biotechnological applications.