The male gametophyte of higher plants represents an excellent system to study gene regulation, cell fate determination and cellular differentiation in plants because of its relative simplicity compared to the sporophyte and its accessibility for cytological and molecular analysis. Unicellular plant microspores are single haploid cells, which can be isolated in large amounts at a defined developmental stage. Microspores cultured in vitro in a rich medium develop into mature pollen grains, which are fertile upon pollination in vivo. It is reported here that isolated Antirrhinum majus microspores when cultured in an optimal medium develop to form mature, fertile pollen. Their development closely resembled that of pollen formed in vivo. Isolated microspores were bombarded with Aquorea victoria Green Fluorescent Protein (GFP), Discosoma Red Fluorescent Protein (dsRFP) and β-glucuronidase (GUS) reporter genes under the control of various promoters and transient expression was observed throughout pollen development in vitro. Bombarded and not bombarded in vitro-matured pollen grains were able to germinate both in vitro and on receptive stigmas and to set seed. The protocol of maturation, transient transformation and germination of Antirrhinum majus pollen in vitro described here provides a valuable tool for basic and applied research.