The goldfish retinal explant system of Landreth and Agranoff was used to study endogenous protein synthesizing activity of retinal ganglion cell axons regenerating in culture. Light and electron microscopic examination of axonal fields showed that axons were free of nonneural cell investment. Decentralized axons were incubated with a mixture of tritiated amino acids, and direct quantitative microanalysis of protein and tritium radioactivity was carried out on individual axonal fields. Our findings showed that radioactive amino acids were incorporated into axonal protein in a manner that inhibited significantly by cycloheximide, but not by chloramphenicol. Decentralized axons appeared to maintain their viability for at least 3-4 h. Axonal fields maintaining their central connections to the explant incorporated 3H-amino acids at an apparent rate that was similar to decentralized axonal fields. Labeled material transported into axonal fields from ganglion cell bodies appeared in significant amounts after a delay of 2-3 h. Fluorographic patterns of axonal proteins after labeling with either 3H-amino acids or [35S]methionine and separated by microelectrophoresis indicated that primarily tubulin and, to a lesser extent, actin were labeled. Our findings indicate that goldfish retinal ganglion cell axons regenerating in vitro exhibit measureable endogenous protein-synthesizing activity.