Two forms of ghrelin, acylated and desacyl, circulate in plasma. Although acylation is thought to be essential for ghrelin biological activities, recent studies have suggested that desacyl ghrelin may also possess biological activity. A new commercial ELISA system has now enabled us to measure plasma levels of each of these two ghrelin forms separately. This assay system directly measures levels using small amounts of plasma. To evaluate the utility of this assay system, we measured the plasma levels of the two forms of ghrelin in healthy volunteers. Although acylated ghrelin levels were equivalent to those measured previously by RIA, desacyl ghrelin levels were lower than those expected from the total ghrelin levels previously determined by RIA. The ratios of acylated to desacyl ghrelin significantly correlated with previously determined acylated, but not desacyl, ghrelin levels. After BMI adjustment, the levels of acylated, but not desacyl, ghrelin plasma levels were higher in female subjects than those in males. Several metabolic and hormonal parameters significantly correlated with either plasma acylated or desacyl ghrelin levels. These findings indicate that separate measurements of the two ghrelin form levels may provide valuable information on their structure, gender differences, and physiological implications.