The present study evaluated and compared the efficiency of the conversion of t11 18:1 and t12 18:1 to their corresponding dienoic acids (c9,tn 18:2) and assessed whether differences due to gender existed in several tissues of rats. Three groups of 4-wk-old male and female rats were fed for 3 wk a diet supplemented with 0, 0.5, or 1% of a trans-octadecenoic acid isomer mixture (tOIM) containing t11 18:1 and t12 18:1 in equal proportion. t11 18:1 and t12 18:1 were incorporated in a tissue-specific manner, and the accrual was significant with increased dietary intake of these trans fatty acid (tFA) isomers. The t12 18:1 isomer was more readily incorporated into the rat tissues than the t11 18:1 isomer. From t11 and t12 18:1, the respective desaturase products, c9,t11 18:2 and c9,t12 18:2, were formed. The calculated conversion rates varied greatly among the tissues of the rats but they were consistently lower for t12 18:1 than for t11 18:1, suggesting that t12 18:1 is a poorer substrate than t11 18:1 for Delta9-desaturase. For both fatty acids investigated, the calculated conversion rates in decreasing order of conversion efficiency were: testes = kidneys > adipose tissue > ovaries > muscle > liver > heart. Overall, there were distinct differences in the conversion of t11 18:1 and t12 18:1, indicating that these 2 fatty acids are metabolized differently despite their structural similarities. Such metabolic differences in tFA accumulation and metabolism may have potential implication in assessing the safety of these tFA isomers because there is a positive correlation between the intake of tFA and the incidence of various diseases.