This chapter describes the initiation of photoautotrophic (PA) cultures of various species of higher plants. It discusses the culture initiation methods, medium requirements, and growth and photosynthetic characteristics. The specific uses of PA cultures are also presented. PA cultures utilize photosynthesis to provide energy and carbon for growth, so no sugar or other energy-producing compounds are added to the culture medium. All PA cultures have CO2compensation concentrations higher than leaves, and this appears to be correlated with much higher dark respiration rates. Most cultures require a relatively long initiation period, except for the N. plumbaginifolia protoplast system, but these cells begin to regenerate plants and do not form a homogeneous cell suspension in liquid medium without sugar. The PA suspension cultures also exhibit the usual advantages of suspension cultures—namely, ease of transfer, direct contact of most cells with the medium, relatively homogeneous cell population, freedom from microorganisms, readily extractable tissue, easily changed medium, and growth in many different vessels, including large fermenters. The PA cultures can be used to study some unique phenomena, such as the reversible change of chloroplasts to amyloplasts and then back to chloroplasts upon growth in the dark with sugar in the medium, followed by growth in the light without sugar, respectively. © 1992 Academic Press Inc.