The mRNA coding for the ammonium inducible NADP-specific glutamate dehydrogenase (NADP-GDH) from Chlorella was studied in induced and uninduced cells to determine the molecular mechanisms which regulate the cellular levels of this enzyme. A procedure for isolation of a high yield of total undegraded cellular polysomes was developed. The crosslinking reagent, dimethyl suberimidate, was employed to prepare a stable NADP-GDH-crosslinked-Sepharose-4B antigen affinity column for the purification of rabbit anti-NADP-GDH IgG. Binding studies with ¹²⁵I-labelled antibody and total polysomes, isolated from induced and uninduced cells, showed that the NADP-GDH was being synthesized on polysomes from both types of cells. When poly(A)- containing RNA was extracted from polysomes isolated from induced and uninduced cells, and translated in an mRNA-dependent in vitro translation system, NADP-GDH antigen was synthesized from the RNA from both sources. Based on sucrose density gradient analysis, Chlorella NADP-GDH mRNA has a sedimentation coefficient of 18 Comparison of the amounts of NADP-GDH synthesized in vitro from poly(A)-containing RNA and non-poly(A)-containing RNA showed the NADP-GDH mRNA contained polyadenylic acid sequence. By use of an indirect immunoadsorption procedure, the NADP-GDH mRNA was purified five- to sevenfold from total poly(A)-containing RNA. The overall purification of the NADP-GDH mRNA from total polysomal RNA was approximately two hundred-fold. Complementary DNA was synthesized from the partially purified RNA with reverse transcriptase. The cDNA sequences hybridized to the least abundant class of mRNA sequences present in total poly(A)-containing RNA. In vitro translation of total poly(A)-containing RNA showed that NADP-GDH synthesis was 0.1% of total protein synthesis. Upon addition of inducer to previously uninduced, synchronous cells, the amount of translatable NADP-GDH mRNA increased in a linear fashion after 30 min of the induction period. A change in rate of NADP-GDH mRNA accumulation was observed after 30 min of the induction period. The results support the prediction that since the NADP-GDH enzyme is unstable in vivo, during periods of NADP-GDH accumulation, the NADP-GDH mRNA accumulates. When poly(A)-containing RNA, isolated from uninduced synchronous cells was translated in vitro, NADP-GDH antigen was synthesized at each time in the cell cycle examined. The amount of translatable NADP-GDH mRNA increased throughout the cell cycle with a rate change occuring during the S-phase. This pattern of NADP-GDH mRNA accumulation is consistent with the hypothesis that NADP-GDH mRNA accumulates in uninduced cells at a rate proportional to gene dosage. These results provide one explanation for the observed pattern of enzyme potential in synchronous cells cultured in the absence of inducer. The data are consistent with the possibility that a single mRNA, which is subject to post-transcriptional modification by the inducer, codes for NADP-GDH.