Nephrocalcinosis develops spontaneously in female rats and is a risk factor for renal disease. Dietary omega-3 polyunsaturated fatty acids (omega-3 PUFAs) may ameliorate the development of nephrocalcinosis, depending on the type and structural form of omega-3 PUFAs present. The objective is to compare diets with omega-3 PUFAs from different sources on modifying renal fatty acid composition and development of nephrocalcinosis. Young (28 d) female Sprague-Dawley rats (n=10/group) were pair-fed 12% fat diets consisting of either corn (CO) or omega-3 PUFAs as flaxseed (FO), menhaden (MO), or krill oil (KO) for 8 weeks. Rats were individually housed in metabolic cages to collect urine. After 8 weeks, the kidneys were dissected and weighed. Kidney fatty acid content was determined using gas chromatography. Kidney calcium and phosphorus content was determined using inductively-coupled plasma spectrometry. Urinary acidity was determined with pH meter. Urinary n-acetyl-ß-D-glucosaminidase (NAG) activity was determined by a colorimetric assay. Creatinine clearance (CrC) was calculated from urinary and serum creatinine concentrations determined using enzymatic assays. Higher ( P <0.05) kidney weights were observed in rats fed KO compared to rats fed MO or FO. The fatty acid composition of the kidneys reflected the diet, with higher omega-3:omega-6 ratios in the rats fed KO, MO, FO compared to rats fed CO. Rats fed KO had the highest ( P =0.01) kidney Ca content, suggesting the development of nephrocalcinosis. Rats fed KO had more acidic urine ( P <0.05) compared to rats fed FO. There was also a tendency ( P =0.07) for increased NAG activity in rats fed KO, a marker for renal injury. There was no apparent change in renal function measures of urinary output ( P =0.90) or CrC ( P =0.70). Different dietary sources of omega-3 PUFAs may influence the development of nephrocalcinosis. Compared to other omega-3 PUFAs, feeding rats KO had higher urinary acidity resulting in kidney injury and Ca deposition.