The aim of this study was to investigate the role of the NO system in ovarian function by determining if Arg supplementation affects cell proliferation in ovarian follicles in nutritionally compromised ewes. Ewes were stratified by weight and randomly assigned into either control (C; n = 35), overfed (O; 2xC; n = 37), or underfed (U; 0.6xC; n = 36) groups 8 weeks before Arg-treatment. Ewes from each nutritional group were randomly assigned to one of 2 i.v. treatments: saline (~10 mL) or Arg (L-Arg-HCl, 155 µmol Arg/kg of BW) which was initiated on d 0 of the estrous cycle and administered 3 times per day (0700, 1400, 2100 h) until ovary collection at the late-luteal stage of the first estrous cycle, or early or mid-luteal stages of the second estrous cycle. Ovaries were fixed in formalin solution followed by immunohistochemical localization of Ki67 (a marker of proliferating cells), and image analysis of granulosa (G) and thecal (T) layers. Data were analyzed by ANOVA using SAS. During nutritional treatment, C maintained BW, O gained 6 ± 1.2 kg, and U lost 14 ± 1.3 kg. Ki67 was detected in G and T layers, and other ovarian compartments. Cell proliferation in G and T of healthy follicles was affected by stage of the estrous cycle, but not plane of nutrition or Arg-treatment. Cell proliferation was 1.3–1.5-fold greater (P < 0.001) at early and mid than late luteal phase. For G and T of healthy follicles, interactions (P < 0.05) between plane of nutrition, Arg-treatment and/or stage were detected, demonstrating stimulatory effects of Arg-treatment on cell proliferation at the early luteal phase in O and U, and inhibitory Arg-effects at the mid-luteal phase in O ewes. Cell proliferation was greater (P < 0.001) in healthy antral than atretic follicles in all groups (14.4 ± 0.4 vs. 1.9 ± 0.1%). These data show that cell proliferation in G and T layers is affected by plane of nutrition, Arg-treatment and/or stage of the estrous cycle that likely affects follicular function. The mechanism of regulation of ovarian cell proliferation by diet and Arg remains to be elucidated. Supported by USDA-AFRI grant 2011–67016–30174, and Hatch Projects ND01748 and ND01754 to ATGB and DAR.