I131-thyroxine and other labeled iodophenols in dilute aqueous buffer solution were applied to filter paper strips for chromatography, and the applied samples were permitted to dry for periods ranging from 0–240 min before the strips were suspended in the chromatography jars. Drying for periods as brief as 10–20 min resulted in a spontaneous loss of I131 from the iodinated compounds and an increase in I131-iodide. The extent of the deiodination was quite variable, but, in most experiments with I131-thyroxine, increases in radioiodide from 3- to 5-fold were observed after 15–30 min of drying. Other I131-iodophenols that were tested (3′,3,5-triiodothyronine, 3′,5′,3-triiodothyronine, 3,3′- diiodothyronine, 3,5-diiodotyrosine, 3-monoiodotyrosine) usually displayed radioiodide increases of 100–300% after 20–30 min of drying. These drying times are not unusual for the preparation of filter paper chromatograms, and lack of awareness of the phenomena described in this report could lead to serious error in biological deiodination studies. I127 measurements revealed an increase in stable iodide as well as in radioiodide during spontaneous deiodination of I131-thyroxine. However, inclusion of 5–20 μg of thyroxine carrier in the I131-thyroxine sample applied to the paper resulted in a reduction in the percentage of I131-iodide released by deiodination. The spontaneous deiodination was completely inhibited if sufficient serum or serum albumin was present, and it was partially inhibited by serotonin and propylthiouracil (2 ×l0-3 M). Inclusion of sufficient ethanol or propylene glycol in the sample applied to the paper markedly reduced the deiodination, but butanol had little, if any, inhibitory effect. Drying in the dark reduced the deiodination only slightly, and no reduction was observed with added iodide, EDTA or methimazole. Short wave ultraviolet light greatly enhanced the spontaneous deiodination. Other than radioiodide, the only I131-component which increased during the course of the reaction was unidentified origin material. This increased only slightly, however, compared with the radioiodide increase. Spontaneous deiodination was observed on a variety of filter papers, and also on other surfaces such as glass paper and thin layer silica gel. It was not observed, however, during paper electrophoresis.