Histopathological studies suggest that the stem cells of human teratomas may be classified into two major categories: nullipotent stem cells, and multipotent stem cells, capable both of self-renewal and differentiation into a wide range of somatic and extraembryonic cell types. We have isolated a multipotent stem cell clone from the human teratoma cell line GCT 27, and compared its properties to a nullipotent clone derived from the same strain. The multipotent clone GCT 27 X-1 gave rise to colonies of mixed cell morphology in vitro. Analysis of cell surface, cytostructural and extracellular matrix markers in GCT 27 X-1 cells showed that the stem cells of this line were very similar in phenotype to nullipotent cells. The two cell clones were predominantly hypotriploid, and contained several marker chromosomes in common. GCT 27 X-1 was feeder-cell-dependent for continuous growth in vitro; removal of the feeder layer resulted in differentiation of the stem cells into a variety of cell types, some with characteristics of extraembryonic endoderm, others showing neuronal properties. When transplanted into nude mice, GCT 27 X-1 cells gave rise to teratocarcinomas containing embryonal carcinoma stem cells, and many other cell types: yolk sac carcinoma cells; cells producing alphafetoprotein or human chorionic gonadotrophin; glandular, columnar, cuboidal, and squamous epithelium; primitive mesenchyme and cartilage; neuroectodermal cells. Nullipotent GCT 27 C-1 cells could form colonies in the absence of feeder layers, but multipotent GCT 27 X-1 cells could not. While a range of known growth factors and related substances failed to substitute for feeder layers in supporting the growth of GCT 27 X-1 stem cells, supernatants from yolk sac carcinoma cell line GCT 44 could partially replace the feeder cell requirement. Thus, the results revealed a basic difference in growth control between these multipotent and nullipotent human embryonal carcinoma cells, and suggested a possible paracrine regulatory pathway between multipotent stem cells and yolk sac carcinoma cells.