The regulation of 25-OH-D3 metabolism in isolated cells of one kidney and microdissected single nephron segments from the contralateral kidney was studied. Preparations from rats fed a normal diet produced only 24,25-OH2-D3, in isolated cells (7.9 pmol/1.25 X 10(6) cells/min; 5 micron 25-OH-D3), in proximal convoluted tubules (PCT; 1.9 pmol/mm/min; 0.1 mM 25-OH-D3), and proximal straight tubules (PST; 1.5 pmol/mm/min; 0.1 mM 25-OH-D3). Distal tubules (DT) did not metabolize 25-OH-D3. Rats pretreated with 1,25-(OH)2-D3 produced more 24,25-(OH)2-D3 in PST (2.49 pmol/mm/min), with no change of metabolism in the isolated cells, PCT and DT. Preparations from vitamin D deficient rats produced both 24,25-(OH)2-D3 and 1,25-(OH)2-D3: 7.2 and 4.6 pmol/1.25 X 10(6) cells/min, respectively, in the cells, 0.9 and 0.3 pmol/mm/min in PCT and 1.2 and 0.4 in PST. These results confirm that rat PCT and PST have 24-hydroxylase activity, that in vitamin D deficiency, rat renal cells, PCT and PST all have both 24-hydroxylase and 1 alpha-hydroxylase activity, and that 1,25-(OH)2-D3 pretreatment increases the 24-hydroxylase activity in the PST but not in the PCT.