Headspace single-drop microextraction has been combined with microvolume UV-vis spectrophotometry for iodine determination. Matrix separation and preconcentration of iodide following in situ volatile iodine generation and extraction into a microdrop of N,N'-dimethylformamide is performed. An exhaustive characterization of the microextraction system and the experimental variables affecting iodine generation from iodide was carried out. The procedure employed consisted of exposing 2.5 microL of N,N'-dimethylformamide to the headspace of a 10 mL acidic (H(2)SO(4) 2 molL(-1)) aqueous solution containing 1.7 molL(-1) Na(2)SO(4) for 7 min. Addition of 1 mL of H(2)O(2) 1 molL(-1) for in situ iodine generation was performed. The limit of detection was determined as 0.69 microgL(-1). The repeatability, expressed as relative standard deviation, was 4.7% (n=6). The calibration working range was from 5 to 200 microgL(-1) (r(2)=0.9991). The large preconcentration factor obtained, ca. 623 in only 7 min, compensate for the 10-fold loss in sensitivity caused by the decreased optical path, which results in improved detection limits as compared to spectrophotometric measurements carried out with conventional sample cells. The method was successfully applied to the determination of iodine in water, pharmaceutical and food samples.