Macromolecular Gd(III) chelates are superior magnetic resonance imaging (MRI) contrast agents for blood pool and tumor imaging. However, their clinical development is limited by the safety concerns related to the slow excretion and long-term gadolinium tissue accumulation. A generation 6 PAMAM Gd(III) chelate conjugate with a cleavable disulfide spacer, PAMAM-G6-cystamine-(Gd-DO3A), was prepared as a biodegradable macromolecular MRI contrast agent with rapid excretion from the body. T₁ and T₂ relaxivities of the contrast agent were 11.6 and 13.3 mM⁻¹sec⁻¹ at 3T, respectively. Blood pool and tumor contrast enhancement of the agent were evaluated in female nude mice bearing MDA-MB-231 human breast carcinoma xenografts with a nondegradable conjugate PAMAM-G6-(Gd-DO3A) as a control. PAMAM-G6-cystamine-(Gd-DO3A) resulted in significant contrast enhancement in the blood for about 5 mins, and Gd-DO3A was released from the conjugate and rapidly excreted via renal filtration after the disulfide spacer was cleaved. The nondegradable control had much longer blood circulation and excreted more slowly from the body. PAMAM-G6-cystamine-(Gd-DO3A) also resulted in more prominent tumor contrast enhancement than the control. However, PAMAM-G6-cystamine-(Gd-DO3A) demonstrated high toxicity due to the intrinsic toxicity of PAMAM dendrimers. In conclusion, although PAMAM-G6-cystamine-(Gd-DO3A) showed some advantages compared with the nondegradable control, PAMAM dendrimers are not suitable carriers for biodegradable macromolecular MRI contrast agents, due to their high toxicity.