Iron-sulfur [Fe-S] clusters are cofactors of proteins involved in electron transfer, enzyme catalysis, radical generation, sulfur donation, and signal transduction. Biogenesis of [Fe-S] clusters is mediated by numerous conserved proteins present in E. coli and in mitochondria of eukaryotic cells such as yeast and humans. Although a completely reconstituted system for study of this process does not yet exist, isolated intact mitochondria are capable of synthesizing new [Fe-S] clusters when supplied with a few ingredients. Here we describe methods for studying the biogenesis of [Fe-S] clusters in intact mitochondria. In these assays, metabolically active mitochondria isolated from a wild-type Saccharomyces cerevisiae strain are incubated with 35S-cysteine. The 35S is rapidly (∼15 min) and efficiently incorporated by physiologic pathways into newly formed [Fe-S] clusters and inserted into target proteins. Proteins labeled with [Fe-35S] clusters are then separated by native polyacrylamide gel electrophoresis followed by autoradiography, thereby allowing direct visualization and quantitation. Both endogenous (Aco1p aconitase) and newly imported (Yah1p ferredoxin) apoproteins can be used as substrates. [Fe-S] cluster biogenesis in isolated intact mitochondria is greatly enhanced by the addition of nucleotides (GTP and ATP) and requires hydrolysis of both. A major advantage of the methods described here is that neither in vivo overexpression of target substrates nor enrichment by immunoprecipitation is necessary to detect radiolabeled proteins. It is also not necessary to perform these assays under anaerobic conditions, because intact mitochondria are capable of protecting newly formed [Fe-S] clusters from oxidative damage.