Spontaneously hypertensive (SH) rats are known to have an increased content of chondroitin sulfate (CS) proteoglycans (PG) in the aorta as compared to normotensive Wistar Kyoto (WKY) rats. In the present study we have compared WKY and SH rat aortas with respect to [35S]sulfate incorporation in vivo and in vitro. The specific activity (cpm/mg aorta) of the total glycosaminoglycan (GAG) pool from SH rat aorta, measured 48 h after intraperitoneal injection of [35S]sulfate, was twice as high as that of WKY aorta GAG. After in vitro incubation of aortas for 4 or 6 h, the specific activity (cpm/mg aorta) of glycosaminoglycans from SH rat was 2.4- to 7.1-fold higher than in controls. Labeled PG were extracted with 4 M guanidine from aortas which had been incubated with [35S]sulfate, and chromatography of the extract on Sepharose CL-6B yielded two incompletely resolved peaks, one emerging with the void volume (peak I) and one in a more retarded position (peak II). Peak I (WKY) contained nearly equal amounts of CS and HS (53 and 46%, respectively) and a small amount of DS (8%). Peak II (WKY) (Kav, 0.34) was divided into two fractions; the fraction of larger molecular weight (II A) contained 43% CS, 35% DS, and 20% HS, whereas the smaller fraction (II B) contained 40% CS, 51% DS, and 5% HS. In each corresponding pool from SH rat aorta, a similar proportion of HS was found, but the DS content was approximately half, and the CS content was correspondingly greater. The estimated molecular weights of the CS/DS chains in peaks I, II A, and II B from WKY aorta were 34,600, 18,800, and 11,600 daltons, respectively, whereas the corresponding values for the SH rat aorta pools were 32,300, 24,700, and 17,000 daltons, respectively. The proportions of 4- and 6-sulfated galactosamine residues as well as the degree of sulfation of the CS/DS PG were similar in the two strains. The HS-PG was larger in the WKY rat aorta and was made up of larger HS chains (Mr 26,600 vs. 16,100); however, the degree of sulfation was apparently similar in the two strains. These results suggest that the rates of PG synthesis and/or degradation and the PG structure are altered in the SH rat aorta.