Abstract: This study was conducted to examine the effects of temperature, time, location and acidification on urinary Ca and N. Urine was collected from 4 horses into a clean bucket and poured through cheesecloth. Urine was stirred and 5 mL was pipetted into vials and tightly capped. Twenty-one different treatments were evaluated with combinations of the following variables: urine held in total collection apparatus (yes or no), acid (none, added immediately, or added after urine was frozen and thawed), time held and temperature until freezing (frozen immediately, 6 h at 25�C, 6 h at 10�C, 12 h at 25�C, or 12 h at 10�C). Sample preparation was completed within 45 min. Acid was added at a rate of 20 �L of 30% Hcl mL ¹ urine. All samples were frozen at -4�C. Urinary Ca was higher when acid was added compared to no acid regardless of holding time, temperature, or location (p<0.01). The addition of acid prior to freezing tended to result in samples having higher Ca than samples with acid added after thawing (p = 0.07). There was no effect of holding time or temperature on Ca (p = 0.86). Urine N was unaffected by the addition of acid to the sample (p=0.22). Urinary N tended to be lower when urine was placed in the total collection apparatus for either 6 or 12 h than when urine was not placed in the apparatus (p = 0.06) and urinary N was higher when acid was added after thawing compared to before freezing (p<0.01). There was no difference in N based upon holding time (p=0.77). In this experiment, all samples were tightly capped and N was not able to volatilize. In the field, samples may sit open to air for a time after collection. Therefore, a portion of the study was repeated to test samples left uncapped. Sample processing was conducted as indicated before except that each holding time and temperature combination was repeated with an uncapped vial. Urine from 4 horses was collected and evaluated with 27 treatments. Five combinations of holding time and temperature were examined (frozen immediately, held for 6 h at 25�C, held for 6 h at 6�C, held for 12 h at 25�C, held for 12 h at 6�C). The same acidification methods as indicated before were used. Urine held uncapped for either 6 or 12 h had a higher N (p<0.01) than samples held for either 6 or 12 h while capped. These data suggest that urinary Ca is more sensitive to the addition of acid than urinary N while N is more sensitive to evaporation; however urine handling methods influence results of both.