We describe a double-label in situ hybridization protocol based on the optimized synthesis of biotin-labeled RNA probes. Biotin-labeled probes are used in conjunction with digoxigenin-labeled probes to simultaneously visualize two different transcripts. One transcript is hybridized with a biotin-labeled RNA probe and visualized as a brown peroxidase reaction product, and the other transcript is hybridized with a digoxigenin-labeled RNA probe and visualized as a blue alkaline phosphatase reaction product. We present several examples in which this double-labeling method has proven useful in determining the spatial and temporal relationships between various transcripts expressed during Drosophila embryogenesis, indicating that this method should be of general use in establishing the relationship of two independent transcription patterns.