Fowlpox virus (FWPV) recombinant vaccines are presently being tested as an antihuman immunodeficiency virus vaccine for humans. However, biosafety, as well as the morphogenesis of FWPV in mammalian cells, are not well understood. Currently, electron microscopy is the method of choice for analyzing virus morphogenesis in cell lines. In this study, four different electron microscopic techniques were used to study FWPV morphogenesis in the Syrian baby hamster kidney (BHK-21) cell line: direct negative stain electron microscopy, ultrathin section transmission electron microscopy, cryoimmunoelectron microscopy, and scanning electron microscopy. The study showed matured viruses, as well as other stages of fowlpox virus maturation, in BHK-21 cells that led to productive virus multiplication. A number of virus-containing vesicles and plasma membrane-associated mature viruses at an early stage in the budding process were observed. In addition, intracellular mature virus was observed in layers of the trans-Golgi network, a characteristic of intracellular mature virus wrapping that results in the formation of intracellular enveloped virus. The size and morphology of FWPV observed in this study are comparable with previously published data. This study presents the first morphological evidence for the release of FWPV by budding in BHK-21 cells.