The secretion of chloride by the rectal gland is an active process. The use of the isolated, perfused rectal gland and pharmacological studies and ion substitution experiments allowed definition of the processes involved in this transport. Measurements of the electrical potential across the gland showed that the initially negative potential became increasingly negative when the gland was stimulated to secrete. Inhibition of NaKATPase with ouabain stopped the secretion of chloride, indicating that NaKATPase was indispensable for the secretion of chloride by the gland. The use of loop diuretics such as furosemide and derivatives and ethacrynic acid that target the chloride cotransporter NKCC1 also inhibited the secretion of chloride, again implying that NKCC1 was necessary for the operation of the system. Thiocyanate or removal of chloride also reduced the secretion of chloride, as did substitution of sodium for other cations or removal of potassium. Thus, the secretion of chloride required working NaKATPase, NKCC1, chloride, sodium, and potassium. Ion substitution experiments showed that chloride binds to its transport pathway at two binding sites. Intracellular electrolyte measurements showed that intracellular chloride drops after stimulation, indicating that stimulation increases a conductance that allows chloride to move out of the cell.