Proteins excreted into the culture medium by carrot cells during somatic embryogenesis in the absence of 2, 4-dichlorophenoxy acetic acid (2, 4-D) and during unorganized cell proliferation in the presence of 2, 4-D were analyzed by SDS polyacrylamide gel electrophoresis (PAGE). During somatic embryogenesis extracellular proteins with apparent molecular masses of 13, 17, 29, 38, and 46 kD were released into the medium. These proteins were reduced or absent in media of 2, 4-D-grown cells. In contrast, cells grown in the presence of 2, 4-D released proteins with apparent molecular masses of 27, 36, 40, 44, 48, and 72 kD. Concanavalin A staining revealed that the 29-, 46-, and 72-kD proteins as well as a doublet of 52/54-kD extracellular proteins were high-mannose type glycoproteins. Carrot cell lines impaired in somatic embryogenesis failed to release one or several of these proteins or produced aberrant forms in the absence of 2, 4-D. Somatic embryogenesis in cell lines missing one or several extracellular proteins were partially restored after addition of extracellular proteins obtained from embryogenic cell lines. In contrast, cell lines producing aberrant forms of extracellular proteins could not be complemented. Somatic embryogenesis could be completely blocked by tunicamycin and deoxynojirimycin. Tunicamycin treatment resulted in the presence of deglycosylated forms of the 46-and 52/54-kD extracellular proteins; deoxynojirimycin treatment resulted in higher molecular mass forms of the 52/54-kD glycoproteins. Somatic embryogenesis in tunicamycin-blocked cultures but not in deoxynojirimycinblocked cultures could be completely rescued by addition of extracellular proteins from untreated embryo cultures. The complementing extracellular proteins were active in a narrow concentration range of 1-3 nM if a single protein was involved. The complementing activity was only found in media from embryogenic cell lines. Microscopical observation of tunicamycin-treated cells revealed that embryogenesis was affected at a very early stage, before globular stage embyros are formed. These results demonstrate that the phytohormone-controlled release of several extraceUular proteins that must be correctly glycosylated for their activity is essential for carrot somatic embryogenesis to occur.