In contrast to mammalian ciliary neurotrophic factors (CNTFs), chick CNTF is secreted, although it lacks an N-terminal signal. We determined that a 52 aa region of chick CNTF containing an internal hydrophobic domain could direct secretion of rat CNTF. Using a stable cell line that overexpressed chick CNTF, we found that chick CNTF immunoreactivity was punctate throughout the cytosol. Cellular fractionation confirmed chick CNTF to be protected by vesicles. Chick CNTF did not colocalize with fibronectin, calreticulin, wheat germ agglutinin binding sites, or with transferrin receptor. The distribution of chick CNTF was altered neither by brefeldin A nor by chloroquine treatment. Although the punctate pattern of chick CNTF immunoreactivity was not due to reuptake, chick CNTF could be found in a cellular compartment labeled after a brief incubation with dextran microbeads. When synthesized in vitro, chick CNTF did not translocate into microsomes. We conclude that chick CNTF is secreted via a nonclassical pathway.