The purpose of this study was mainly to examine some basic problems of the Hanssen technique for the evaluation of single nephron glomerular filtration rate (SNGFR). Firstly, experiments were performed using Wistar rats to determine the amount of C14-ferrocyanide necessary to give sufficient C14 radioactivity in dissected nephrons. Using 200 muCi of C14-ferrocyanide, the samples of dissected nephrons provided only twice as much as background. Hence, we chose the amount of 600 to 700 muCi of C14-ferrocyanide for each rat in the later experiments, which yielded the radioactivity 5 to 6 times as much as background. Secondly, the homogeneity of intrarenal C14-ferrocyanide distribution was tested. Five superficial (SUP) and juxtamedullary (JM) proximal tubules four different regions were dissected and the radioactivity of those nephrons was counted. The distribution of C14-ferrocyanide in each piece was representative of the overal distribution. The summary of morphologic and clearance studies were as follows. The mean lengths of proximal tubules of 40 SUP nephrons, 5.3 +/- 0.2 mm, and 39 JM nephrons, 6.3 +/- 0.1 mm, were significantly different (P less than 0.01). The mean glomerular diameter of the JM nephrons, 110 +/- 2.7 mu, was significantly greater than that of the SUP nephrons, 95 +/- 3.1 mu (P less than 0.01). The mean SNGFR of 129 SUP nephrons, 23.4 +/- 4.2 nl/min/100g Body Weight (BW), and that of 130 JM nephrons, 34.5 +/- 5.1 nl/min/100g BW, were significantly different (P less than 0.01). The ratio of superficial/juxtamedullary nephron glomerular filtration rate (S/JM Ratio) was 0.85 +/- 0.02. We conclude that C14-ferrocyanide is regularly distributed to the kidney and that it is necessary to infuse sufficient amount of C14-ferrocyanide in this technique.