The aim of the present study was to investigate changes in nitrate (NO3 - ), progesterone (P4), and estradiol-17β (E2) concentrations in follicular fluid (FF) according to the phases of the estrous cycle and to follicle quality and size in cattle. Pairs of ovaries were classified according to the morphological characteristics of the corpus luteum (CL) as mid-luteal or follicular phase. The follicles were classified in three groups according to size: small (<5 mm, n = 120), medium (5 to 8 mm, n = 82), and large (>8 mm, n = 120). Small and medium follicles (n = 2-3) with a clear surface and from the same ovary were punctured and their wall gently scraped. The aspirated FF and granulosa cells (GCs) were pooled separately. Large follicles (high vascularity in theca cells and clear FF) were dissected and FF and GCs collected separately. Follicle integrity was verified by GC-DNA electrophoretic analysis. Follicles were classified as atretic (DNA fragments in multiples of 185 bp) or healthy (without DNA fragments). Concentration of NO3 - was estimated by chemiluminescence and P4 and E2 by radioimmunoassay. The greatest NO3 - concentration was observed (P < 0.05) in medium atretic follicles. Progesterone concentration increased (P < 0.05) in atretic follicles according to size (P < 0.05) and was greater in atretic than in healthy follicles. Progesterone was also greater (P < 0.05) in atretic follicles of the same size collected during the mid-luteal phase compared to the follicular phase. However, 15.5% of follicles that did not show DNA fragmentation had an E2:P4 ratio <1. Estradiol-17β concentration increased (P < 0.05) in healthy and atretic follicles according to size and was greater (P < 0.05) in healthy than in atretic follicles. These results suggest that: 1) NO3 - did not change with follicular development, except in medium atretic follicles suggesting a possible role in follicular atresia of subordinate follicles and 2) changes in steroids concentrations may precede DNA fragmentation.