The petal is an important floral organ of higher plants. To study the mechanism of petal development, the in vitro regeneration system of petals was established in Petunia. High-frequency induction of petals occurred directly from explants on the media containing the combination of N6-benzyladenine (6-BA) and indole-3-acetic acid (IAA). Expression analysis of genes involved in flower development indicated that these genes were classified into three types. ABERRANT LEAF AND FLOWER (ALF) gene was induced during petal regeneration. Whereas, B-class and E-class genes, and genes involved in cell division were constitutively upregulated. In contrast, C-class and D-class genes were not expressed in explants and regenerated tissues. Further, in situ hybridization analysis showed that both ALF and GREEN PETAL (GP) expression were spatially regulated. The results suggest that differential regulation of gene expression occurs in the presence of hormones during petal regeneration, and hormone-regulated gene expression might be required for petal regeneration. This study provides the preliminary information to understand the mechanism of petal regeneration.