Mature pollen of lodgepole pine (PinusconcortaDougl.), yellow cypress (Chamaecyparisnootkatensis(D. Don) Spach), western hemlock (Tsugaheterophylla(Raf.) Sarg.), jack pine (PinusbanksianaLamb.), and black spruce (Piceamariana(Mill.) B.S.P.) was bombarded with gold particles coated with four different plasmid constructions, pRT99GUS, pBM113Kp, pAct1-D, and pGA984, using the biolistic PDS-1000/He device. A protocol was devised for efficient gene transfer and gene expression assay in pollen. False positive results for expression of the β-glucuronidase (GUS) gene assayed with the substrate X-glucuronide were observed with pollen of yellow cypress, western hemlock, and lodgepole pine. The highest levels of transient GUS gene expression were obtained with plasmid pBM113Kp, which carried the GUS gene under the control of the wheat abscisic acid inducible early methionine promoter. The plasmids pRT99GUS (35S promoter) and pAct1-D (rice actin promoter) yielded similar intermediate levels of transient GUS gene expression. The pollen-specific promoter of the α-tubulin gene fromArabidopsisthaliana(pGA984) yielded the lowest levels of gene expression in pollen. Of the four species, yellow cypress showed the lowest levels of transient GUS gene expression and black spruce yielded the highest levels. The neomycin phosphotransferase II (NPT II) gene was also tested as a reporter gene for pollen transformation and was easily assayed via ELISA. The fusion gene between NPT II and GUS genes was detected at a lower level than the nonfused NPT II gene when under the control of the same 35S promoter. The method devised here could be used for the study of tissue-specific gene expression in conifer pollen.