The relationship between prior knowledge of and interest in a topic is complex. Although knowledge and interest may often go hand in hand, they do not necessarily correlate. The purpose of this study was to separate the effects of prior knowledge and topic interest on second language reading comprehension. Participants in the study were 104 students of English as a second language in an English-for-academic-purposes program at a large American university. On the basis of a prior-knowledge test and a topic-interest inventory, each student read passages and took multiple-choice comprehension tests on topics for which they had all four possible combinations of high and low topic interest and high and low prior knowledge. Results on the reading comprehension measure for the two main effects of prior knowledge and topic interest, although in the expected direction, did not reach significance, possibly because of a significant interaction between those two variables. There was also a significant effect for English proficiency level, as well as a significant interaction between interest and gender, with males more influenced by high topic interest than females. Our results are compared to those of other first and second language studies that have explicitly investigated the interrelationships among topic interest, prior knowledge, and gender.