Optimal conditions for protein synthesis in incubated slices of rat brain were determined to be: thickness 0.3 mm under air (for newborn 0.4-0.6 mm) or 0.5 mm under oxygen; temperature, 35-36 degrees C; hepes (N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid) buffer; K+, 6-8 mM; Ca2+, 2-3 mM. Though maximum incorporation was found with a Na+ concentration of 110-12- mM. Though maximum incorporation was found with a Na+ concentration of 110-120mM, this requirement appears to be partly osmotic. The Na+ concentration may be reduced to 80 mM without inhibition of incorporation provided adjustment is made for osmotic balance. Mg2+. Optimal pH was 7.2-7.4. The rate of protein synthesis in this medium is 0.08-0.09% replacement of the protein amino acid/h in slices from adults and 1.6%/h in slices from 3-day-old rats. Thus slices from adults synthesize protein at 10-20% of the in vivo rate whereas slices at 3-day-old brainincorporate amino acid at 70-80% of the in vivo rate for young rats.