The present study was undertaken to ascertain the transfer process of total cholesterol and its fractions (HDL-C, LDL-C, Free-C and esterified-C) from blood into various compartments (Follicular Wall-FW; Granulosa Cells – GC; Follicular Fluid – FF) of the buffalo ovarian follicles across the “blood-follicle barrier” and also to correlate the potential role of the cholesterol and its fractions as a substrate for steroidogenesis in non-atretic and early atretic buffalo ovarian follicles. The FW, GC and FF from non-atretic and early-atretic follicles of pre-ovulatory and ovulatory stages of buffalo ovaries were collected and pooled category-wise. From these samples lipids were extracted as per the standard protocol. The total cholesterol, HDL-C and LDL-C present in these samples was estimated spectrophotometrically, while the free-C and esterified-C were initially separated by partition chromatography and then estimated spectrophotometrically. The concentration of total cholesterol and its fractions decreased progressively from FW to GC, followed by FF, which indicate that all these fractions originate from serum only. Within the health (non-atretic and atretic) status of the follicles, their concentration increased from pre-ovulatory to ovulatory stage, indicating a higher steroidogenic potential and an increase in the number of steroidogenic cells within the follicles, as they grow. Other observations include: higher concentration of total cholesterol and HDL-C in the GC and FF than the FW of early-atretic follicles; higher free-C in all the compartments of early atretic follicles. From these results it is evident that there is sufficient supply of the substrate (total-C and HDL-C) to the thecal cells (FW) for the testosterone synthesis even at the onset of atresia, but the limitation is the availability of the free-C in the GC of the atretic follicles. This may be the cause for the switch over in steroidogenic pattern i.e. from estradiol to progesterone synthesis in atretic follicles.