To address basic cell biology questions such as “Can cells respond to a gravity stimulus?”, a Cell Culture Unit (CCU) is being developed for use in the International Space Station. The CCU will accommodate diverse specimen types (animal, plant, and microbial cells; tissues; aquatic organisms) in up to twenty-four cell specimen chambers. The environmental conditions (e.g. temperature, pH, gas concentrations) will be maintained by medium recirculation and renewal, and gas and heat exchange. The CCU will also provide for the addition of experimental agents (e.g. growth factors), automated sampling, and monitoring of the specimens by video microscopy. Microgravity experiments will be performed using the CCU within the Habitat Holding Rack (HHR); a CCU within the Space Station Centrifuge will serve as an on-board unit-gravity control.The CCU design includes a Cell Specimen Environment Assembly to maintain the specimens, a Video Microscope System to obtain specimen images, an Electronics Assembly to control the subsystems and provide signal conditioning, a Power Supply Subsystem to condition power from the HHR or Centrifuge, an Active Cooling Subsystem to maintain specimen temperature, and a Structural Containment Assembly to house the subsystems and interface with the HHR or Centrifuge. Six reference specimens, encompassing the candidates that will likely be flown and that exercise the required ranges of experimental conditions, were selected to test the individual subsystems and the CCU system. Here we report the design and development testing of the first CCU prototypes (e.g. biocompatibility, medium flow and mixing, gas exchange, sampling, video images, cell and tissue culture). Detailed design and testing will continue towards a first launch of the CCU in 2001.