Over the five years of organizing the HCA meeting, we have observed the technology mature and its adoption spread into many areas of compound screening/evaluation and functional analysis. The Sixth Annual High-Content Analysis 2009 meeting will focus on the next steps of technology development, including new assays and probes, more advanced image analysis and data management, and novel biological models for screening. We will also cover case studies and applications in compound screening, cytotoxicity evaluation, functional analysis, and more.