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The purpose of this study was to determine whether exercise and/or acetaminophen (APAP) alter collagen and cross-linking in the rat gastrocnemius muscle, soleus muscle, and heart. Male Wistar rats (n = 50; 8 wk old) were divided into placebo (PLA) or APAP groups and sedentary (SED) or exercised (RUN) groups. APAP (200 mg/kg) was administered daily by oral gavage. Exercised groups ran on a treadmill 5 days/wk for 8 wk with progression to 60 min/day, 20 m/min, and 8° incline. Tissues were assayed for collagen (hydroxyproline) and hydroxylyslpyridinoline (HP) and lysylpyridinoline (LP) cross-links by HPLC. Collagen content (μg/mg dry weight) was greater in both the gastrocnemius (SED-PLA: 114 ± 16 vs., Run-Pla: 244 ± 32; P < 0.001) and soleus (SED-PLA: 51 ± 7 vs., Run-Pla: 99 ± 27; P = 0.005) of exercised animals. In contrast, collagen content was not significantly greater in exercised animals treated with APAP (SED-APAP: 113 ± 16 vs., Run-Apap: 145 ± 21) and soleus (SED-APAP: 55 ± 8 vs., Run-Apap: 57 ± 10). HP cross-linking (mmol/mol collagen) in the gastrocnemius (SED-PLA: 126 ± 28, RUN-PLA: 50 ± 7, SED-APAP: 41 ± 7, and, Run-Apap: 30 ± 4) and soleus muscles (SED-PLA: 547 ± 107, RUN-PLA: 318 ± 92, SED-APAP: 247 ± 64, and, Run-Apap: 120 ± 17) was lower in exercised rats compared with sedentary rats (P < 0.05). Cross-linking was further reduced in animals treated with APAP (P < 0.05). Neither heart collagen nor cross-linking was influenced by exercise or APAP (P > 0.05). Our findings suggest that exercise and APAP have tissue-specific effects on muscle collagen. Given the widespread use of APAP as an analgesic and antipyretic, further work in humans is warranted., (Copyright © 2015 the American Physiological Society.)