In vitro clonal analyses were performed to gain insight into the mechanisms that control development of neural crest-derived cardiac outflow tract mesenchyme of quail embryos. The cardiac neural crest originates from the posterior rhombencephalic neural tube. The cells leave the neural tube and migrate through the posterior visceral arches to the outflow tract of the heart, where they form the conotruncal ridges. Mesenchyme cells derived from the neural tube from somitic levels 1-3 contained 5 types of clone-forming cells. One class of clones contained up to 6 phenotypes; smooth muscle cells, connective tissue cells, chondrocytes, sensory neuron precursors, serotonergic (putative enteric) neurons, and pigment cells. Another type of clone was totally pigmented, containing melanocytes only, whereas a third type consisted entirely of smooth muscle cells. The remaining classes of clones contained 3 and 4 phenotypes, respectively. Subsequently, mesenchymal cells obtained from posterior visceral arches were cloned in vitro. The major observations from these experiments are the following. 1) The cells have lost the capacity to form sensory neurons. 2) The capacity to form pigment cells is lost as well. 3) Four types of morphologically distinguishable clones were found. The frequency of one type of clone that contains ectomesenchymal cells only (smooth muscle cells, connective tissue cells, and chondrocytes) increased from zero at Hamburger and Hamilton stage 19+/20 to 67% at stage 24, possibly giving rise to visceral arch-derived structures. The frequency of the other 3 types of clones decreased with progressing embryonic development, indicating that these clone-forming cells either pass through the visceral arches, and/or are being converted to cells with fewer developmental potentials.(ABSTRACT TRUNCATED AT 250 WORDS)