Thyrotropin-releasing hormone (TRH), pGlu-His-ProNH2, binds within 1 h to specific receptors on the GH3 strain of pituitary cells. When GH3 cells were incubated for 2 days with 3 nM [2,3-3H-Pro]TRH, an increasing fraction of the total cellular radioactivity (7% after 1 h, 81% after 43 h) was associated covalently with proteins as determined by dialysis, acid precipitation, and gel filtration; this fraction corresponded to label which could not be displaced from intact GH3 cells by the addition of excess unlabeled TRH. R5 and GH12C1 cells, strains which lack TRH receptors, accumulated 16 or 23%, respectively, as much label from [2,3-3H-Pro]TRH as did GH3 cells in 24 h. After 24 h of incubation with [2,3-3H-Pro]TRH and [14C-His]TRH, the ratio of 14C/3H in GH3 cells was the same as in the culture medium, indicating that the intact tripeptide was taken up by the cells. After 24-48 h of incubation with [2,3-3H-Pro]TRH, GH3 proteins appeared to be labeled randomly as surmised by fractionation on Sephadex G-100, DEAE cellulose, Sepharose 4B and sucrose density gradients. In cultures treated with cycloheximide (10 mug/ml) or proline (6.3 mM) the initial binding of [2,3-3H-Pro]TRH to receptors, measured after 1 h, was 97% or 102% of control. However, the incorporation of label from [2,3-3H-Pro]TRH into an acid-precipitable product after 22 h was inhibited by 81 and 74% by cycloheximide (1 mug/ml) and proline (2.5 mM). Formation of [2,3-3H] proline from [2,3-?3H-Pro] TRH was demonstrated by thin layer chromatography; the percentage of non-protein radioactivity with an Rf of proline increased from 20 to 80% in GH3 cells incubated 1 or 24 h with [2,3-3H-Pro]TRH. We conclude that after binding to receptors on GH3 cells, TRH is slowly metabolized to its constituent amino acids, and the products [2,3-3H]proline or [14C]histidine are incorporated into newly synthesized proteins.