Inhibition of aromatase has been an attractive approach for examining the roles of estrogen biosynthesis in various physiological or pathological processes. Effective aromatase inhibitors can serve as potential therapeutic agents for controlling estrogen-dependent diseases such as hormone-dependent breast cancer. Investigations on the development of aromatase inhibitors have therefore expanded greatly in the past two decades. Numerous steroidal agents have been developed that have high affinities for the aromatase enzyme complex and exhibit either competitive inhibition, irreversible inhibition, or mechanism-based (enzyme-activated) inhibition. Mechanism-based inhibitors have distinct advantages in drug design, since these inhibitors are highly enzyme specific, produce prolonged inhibition, and exhibit minimal toxicities. Examination of the structure-activity relationships of the numerous steroidal aromatase inhibitors suggest that the spacial requirements for interaction of agents with the active site of aromatase are very restrictive, permitting only small structural changes to be made on the A-ring and at C-19. Incorporation of small polar substituents at the C-4 position, such as a hydroxyl group, or addition of aryl functionalities at the 7 alpha-position of the steroid, are the exceptions, and inhibitors with such modifications exhibit enhanced affinity for the enzyme. Future investigations of steroidal aromatase inhibitors as probes of the active site of purified aromatase will provide valuable information on enzyme structure at the molecular level, will permit a more detailed examination of the mechanisms of inhibition, and will enhance the development of more specific and effective inhibitors for the treatment of estrogen-dependent breast cancer.