We previously observed that the perichondrium (PC) and the periosteum (PO) negatively regulate endochondral cartilage growth through secreted factors. Conditioned medium from cultures of PC and PO cells when mixed (PC/PO‐conditioned medium) and tested on organ cultures of embryonic chicken tibiotarsi from which the PC and PO have been removed (PC/PO‐free cultures) effect negative regulation of growth. Of potential importance, this regulation compensates precisely for removal of the PC and PO, thus mimicking the regulation effected by these tissues in vivo. We have now examined whether two known negative regulators of cartilage growth (retinoic acid [RA] and transforming growth factor‐β1 [TGF‐β1]) act in a manner consistent with this PC/PO‐mediated regulation. The results suggest that RA and TGF‐β1, per se, are not the regulators in the PC/PO‐conditioned medium. Instead, they show that these two factors each act in regulating cartilage growth through an additional, previously undescribed, negative regulatory mechanism(s) involving the perichondrium. When cultures of perichondrial cells (but not periosteal cells) are treated with either agent, they secrete secondary regulatory factors into their conditioned medium, the action of which is to effect precise negative regulation of cartilage growth when tested on the PC/PO‐free organ cultures. This negative regulation through the perichondrium is the only activity detected with TGF‐β1. Whereas, RA shows additional regulation on the cartilage itself. However, this regulation by RA is not “precise” in that it produces abnormally shortened cartilages. Overall, the precise regulation of cartilage growth effected by the action of the perichondrial‐derived factor(s) elicited from the perichondrial cells by treatment with either RA or TGF‐β1, when combined with our previous results showing similar—yet clearly different—“precise” regulation by the PC/PO‐conditioned medium suggests the existence of multiple mechanisms involving the perichondrium, possibly interrelated or redundant, to ensure the proper growth of endochondral skeletal elements. © 2002 Wiley‐Liss, Inc.