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Events occurring during meiotic prophase I are critical for the successful production of haploid gametes. Many prophase I events are mediated by a meiosis-specific structure called the synaptonemal complex. To date, the limited knowledge we have about the dynamics of these prophase I events in mice comes from fixed, two-dimensional preparations of meiotic cells making it impossible to study the three-dimensional (3D) arrangement of meiotic chromosomes. The current study involves the development of an imaging system to view prophase I events in live mammalian spermatocytes by generating a transgenic mouse, Sycp3–Eyfp21HC, expressing a fluorescently tagged synaptonemal complex protein, SYCP3. Using this live imaging system, the 3D structural arrangement of chromosomes in the different prophase I substages has been characterized in live spermatocytes, and aspects of the 3D architecture of spermatocytes have been observed that would not be possible with existing techniques. Additionally, chromosome movement in prophase I spermatocytes and meiotic progression from pachynema to diplonema were observed following treatment with the phosphatase inhibitor, okadaic acid (OA), which accelerates the progression of cells through late prophase I. These studies demonstrate that the Sycp3–Eyfp21HC live imaging system is a useful tool for the study of mammalian prophase I dynamics.