Cultural systems developed for the successful production of haploid plants from anther and microspore culture of a number of species are lethal to cotton microspores. Components of these systems were examined individually and in simple combinations to determine their specific effect on cotton microspore viability during isolation and culture. An organically buffered pH of 7.0 was critical for survival. The addition of potassium salts, near standard concentrations used for other species, enhanced survival and the cytoplasmic appearance of isolated microspores. Severe toxicity resulted from ammonium, calcium and magnesium salts, and these ions were tolerated only at very low concentrations. Iron, glutamine, serine, inositol, vitamins, and trace minerals were generally not detrimental to microspores at standard concentrations. An isolation and cultural maintenance system was developed that yields large quantities of healthy, viable cotton microspores. This initial step allows for further research in inducing cotton microspores to divide and undergo embryogenesis.