The potential role of cyclic nucleotides in rat testicular development was evaluated by measuring tissue levels of cyclic AMP and cyclic GMP during a time of rapid testicular growth. The levels of the nucleotides were correlated with their distribution in various cell types as well as intracellularly by an immunocytochemical technique. The concentration of cyclic AMP was high during the infantile phase, fell steadily to a nadir prior to puberty and then increased through sexual maturation. Cyclic GMP levels were also elevated during the infantile phase and decreased steadily through sexual maturation. Early in development the distribution patterns for the two cyclic nucleotides were quite similar with fluorescence in almost all cell types. With maturation, cyclic AMP was localized within tubules, primarily in Sertoli cells and spermatogenia, and was observed in cells in the intertubular area. In the mature testis, cyclic GMP fluorescence was associated with cells representative of most stages of spermatogenesis. Cyclic GMP was localized in nuclear elements of many cell types: in nucleoli of Sertoli cells, spermatogonia and of cells in the intertubular area. Staining was also observed along pachytene chromosomes and in a reticular nuclear pattern in a number of cell types. Cyclic AMP was seen in nuclear elements of certain cells, particularly early in testicular development, but the staining pattern was more indistinct than that of cyclic GMP. The distribution patterns of nucleotides at several stages of testicular development suggest that these compounds may be associated with cellular events related to hormone-dependent maturation of the testis.