The requirements for monocytes in lymphocyte proliferation were studied in ultra-microcultures. For this purpose, an ultra-microtest plate was developed which comprises 121 wells, each having 0.23 microliter volume and 0.28 mm2 culture area. Human peripheral lymphocytes were seeded in the wells in numbers ranging from 1-57 cells/well and subsequently stimulated with phytohemagglutinin (PHA). Proliferation, assessed by microscopy in situ, was established in 46% of the wells where adherent non-specific, esterase-positive cells were present and in 6% where such cells were absent. The results indicate that PHA-stimulated human lymphocytes can proliferate in the absence of monocytes. The new microplate should be a valuable tool for dissecting the early events in T cell activation, especially if combined with various analytical methods such as time-lapse video, autoradiography and surface-marker techniques.